Antibody peptide conjugates that have agonist activity at both the glucagon and glucagon-like peptide 1 receptors

ABSTRACT

Described are antibody peptide conjugates (APCs) comprising an antibody conjugated to a peptide analog of glucagon, which have been modified to be resistant to cleavage and inactivation by dipeptidyl peptidase IV (DPP-IV) and to increase in vivo half-life of the peptide analog while enabling the peptide analog to have agonist activity at the glucagon (GCG) receptor and the glucagon-like peptide 1 (GLP-1) receptor and the use of such APCs for treatment of metabolic disorders such as diabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and obesity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application of PCT/US2016/055265filed Oct. 4, 2016, and claims benefit under 35 U.S.C. 119(e) of U.S.Provisional Application No. 62/237,009, filed Oct. 5, 2015, both ofwhich are incorporated herein in their entities.

BACKGROUND OF THE INVENTION (1) Field of the Invention

The present invention relates to antibody peptide conjugates (APCs)comprising an antibody conjugated to a peptide analog of glucagon, whichhave been modified to be resistant to cleavage and inactivation bydipeptidyl peptidase IV (DPP-IV) and to increase in vivo half-life ofthe peptide analog while enabling the peptide analog to have agonistactivity at the glucagon (GCG) receptor and the glucagon-like peptide 1(GLP-1) receptor and the use of such APCs for treatment of metabolicdisorders such as diabetes, non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), and obesity.

(2) Description of Related Art

Pre-proglucagon is a 158 amino acid precursor polypeptide that isprocessed in different tissues to form a number of differentproglucagon-derived peptides, including glucagon, glucagon-likepeptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin(OXM), that are involved in a wide variety of physiological functions,including glucose homeostasis, insulin secretion, gastric emptying, andintestinal growth, as well as the regulation of food intake. Glucagon isa 29-amino acid peptide that corresponds to amino acids 33 through 61 ofpre-proglucagon, while GLP-1 is produced as a 37-amino acid peptide thatcorresponds to amino acids 72 through 108 of pre-proglucagon. GLP-1(7-36) amide or GLP-1 (7-37) acid are biologically potent forms ofGLP-1, that demonstrate essentially equivalent activity at the GLP-1receptor.

During hypoglycemia, when blood glucose levels drop below normal,glucagon signals the liver to break down glycogen and release glucose,causing blood glucose levels to rise toward a normal level. Hypoglycemiais a common side effect of insulin therapy in patients withhyperglycemia (elevated blood glucose levels) due to diabetes. Thus,glucagon's most recognized role in glucose regulation is to counteractthe action of insulin and maintain blood glucose levels.

GLP-1 has different biological activities compared to glucagon. Itsactions include stimulation of insulin synthesis and secretion,inhibition of glucagon secretion, and inhibition of food intake. GLP-1has been shown to reduce hyperglycemia in diabetics. Exendin-4, apeptide from lizard venom that shares about 50% amino acid identity withGLP-1, activates the GLP-1 receptor and likewise has been shown toreduce hyperglycemia in diabetics. There is also evidence that GLP-1 andexendin-4 may reduce food intake and promote weight loss, an effect thatwould be beneficial not only for diabetics but also for patientssuffering from obesity. Patients with obesity have a higher risk ofdiabetes, hypertension, hyperlipidemia, cardiovascular disease, andmusculoskeletal diseases.

Glucagon is a peptide hormone structurally related to GLP-1 that is wellrecognized for its acute ability to increase blood glucose throughstimulation of glycogenolysis and gluconeogenesis (Jiang & Zhang, Am. J.Physio.l Endocrinol. Metab. 284: E671-E678 (2003)). Of lesserappreciation are the chronic effects of glucagon pharmacologycharacterized by increases in thermogenesis, satiety, lipolysis, fattyacid oxidation, and ketogenesis (Habegger et al., Nat. Rev. Endocrinol.6: 689-697 (2010)). Repeated administration of glucagon was firstreported decades ago to yield improvements in rodent metabolism,accompanied with lower body weight (Salter, Am. J. Clin. Nutr. 8:535-539 (1960)). Nonetheless, the inherent risk of hyperglycemia,especially in insulin resistant states such T2DM, has complicated thetranslation of these observations to human study.

The hormone oxyntomodulin (OXM, glucagon-37) is a posttranslationalproduct of preproglucagon processing in the intestine and centralnervous system (CNS) and is secreted from L-cells in the gut in responseto food intake. Discovered in 1983, OXM has been implicated in theregulation of food intake and energy expenditure (Jarrouse et al.,Endocrinol. 115: 102-105 (1984); Schjoldager et al., Eur. J. Clin.Invest., 18: 499-503 (1988)). Central or peripheral administration ofOXM in rats causes a decrease in short term food intake with minimaleffects on gastric emptying (Dakin et al. Endocrinology, 142: 4244-4250(2001), Dakin et al. Endocrinology, 145: 2687-2695 (2004)). Repeatedintracerebroventricular administration of OXM in rats results inelevated core temperatures and reduced weight gain compared to pair-fedanimals, suggesting effects on both caloric intake and energyexpenditure (Dakin et al. Am. J. Physiol. Endocrinol. Metab., 283:E1173-E1177 (2002)).

In related studies, peripheral administration of OXM dose-dependentlyinhibited both fast-induced and dark phase food intake, but unlikeGLP-1, had no effect on gastric emptying. OXM also reduced levels offasting ghrelin and increased c-fos immunoreactivity, in the arcuatenucleus (ARC). Repeated seven-day IP administration of OXM caused areduction in the rate of body weight gain and adiposity in rats (SeeDakin et al. Endocrinology, 145: 2687-2695 (2004)).

Studies of OXM action in mice have demonstrated that although OXM canactivate both the glucagon (GCG) and the GLP-1 receptors, the anorecticactions of OXM require only the GLP-1 receptor, as icy OXM inhibits foodintake in glucagon receptor knockout mice. However, the anorecticeffects of OXM are completely absent in GLP-1 receptor knockout mice.Furthermore, exendin-4, but not OXM, regulates energy expenditure inmice. Hence, OXM appears to be a weak agonist at the GLP-1 receptor,when used in pharmacological concentrations (See Baggio et al.,Gastroenterol. 127: 546-58 (2004)). OXM was also found to ameliorateglucose intolerance in mice fed a high fat diet (Dakin et al., Am. J.Physiol. Endocrinol. Metab. 294: E142-E147 (2008) and increase theintrinsic heart rate in mice independent of the GLP-1 receptor (Sowdenet al., Am. J. Physiol. Regul. Integr. Comp. Physiol. 292: R962-R970(2007). OXM has also been shown to differentially affect GLP-1 receptorbeta-arrestin recruitment and signaling through Galpha (Jorgensen etal., J. Pharma. Exp. Therapeut. 322: 148-154 (2007)) and todifferentially affect hypothalamic neuronal activation followingperipheral injection of OXM (Choudhri et al., Biochem. Biophys. Res.Commun. 350: 298-306 (2006)).

In humans, a single 90 minute intravenous infusion of OXM in normalweight healthy subjects reduced hunger scores and food intake at abuffet meal by about 19%. Cumulative twelve-hour caloric intake wasreduced by about 11% with no reports of nausea or changes in foodpalatability (Cohen et al., J. Clin. Endocrinol. Metab., 88: 4696-4701(2003); Lykkegaard et al., ADA Scientific Sessions, Abstract #1506-P(2003)). More recently, pre-prandial injections of OXM over a four-weekperiod in obese healthy volunteers (BMI about 33) led to a significantreduction of caloric intake on the first day of treatment (about 25%)that was maintained over the course of the study (35% reduction afterfour weeks) (Wynne et al., Diabetes 54: 2390-2395 (2005)). Robust weightloss was observed at the end of the study in treated subjects (1.9%,placebo-corrected). Plasma levels of OXM were similar to that observedin the infusion study (peak concentration about 950 pM). The absence ofany tachyphylaxis and a low incidence of mild and transient nausea(about 3%) despite the relatively high doses necessitated by the poor invivo stability of OXM (plasma t½<12 minutes) renders this hormone one ofthe few obesity targets with both human validation and an attractivetolerability profile.

OXM has a very short half-life and is rapidly inactivated by the cellsurface dipeptidyl peptidase IV (DPP-IV) (Zhu et al., J. Biol. Chem.278: 22418-22423 (2002). However, DPP-IV inhibitors are weight-neutralin the clinic, suggesting that supraphysiological levels of OXM(900-1000 pM) may be required to achieve weight loss in humans. OXMpeptide analogs for inducing weight loss in humans have been the objectof Published International Application Nos. WO03/022304, WO2004/062685,WO2006/134340, and WO2010/096052.

Recently, two independent and simultaneous papers reported the use ofrelatively balanced GLP-1 receptor/GCG receptor co-agonists as being ofenhanced efficacy and safety relative to pure GLP1R agonists in thetreatment of rodent obesity, with simultaneous improvement in glycemiccontrol (Day et al., Nat. Chem. Biol. 5: 749-757 (2009); Pocai eta al.,Diabetes 58: 2258-2266 (2009)). Of related significance is work withoxyntomodulin (OXM), an endogenous precursor to glucagon, which issecreted postprandially by L-cells of the jejuno-ileum together withGLP-1 (Hoist, Regul. Pept. 93: 45-51 (2000); Drucker, Nat. Clin. Pract.Endocrinol. Metab. 1: 22-31 (2005).

Glucagon peptide analogs and derivatives modified to have variousdegrees of activity at the GLP-1 receptor and GCG receptor have beendisclosed in Published International Application Nos. WO2008/1010017,WO2009/155258, WO2011/075393, WO2012/177444, and WO2012/177443. Some ofthe disclosed glucagon peptide analogs were reported therein to haveactivity at both the GLP-1 receptor and GCG receptor; however, thereremains a need for co-agonist peptides that have activity or potency atthe GLP-1 receptor and GCG receptor.

BRIEF SUMMARY OF THE INVENTION

The invention provides antibody peptide conjugates (APCs) that areagonists of the glucagon (GCG) receptor and the glucagon-like peptide 1(GLP-1) receptors. Native glucagon normally has about 1% of the activityof native GLP-1 at the GLP-1 receptor. However, the GCG receptor/GLP-1receptor co-agonist peptides of the invention have activity or potencyat the GCG receptor and GLP-1 receptor. In particular aspects, thepeptides have an EC₅₀ of about 0.007 to about 10.0 nM at the GCGreceptor and an EC₅₀ of about 0.002 to about 10.0 nM at the GLP-1receptor. The peptides herein are useful for the treatment of metabolicdisorders, such as but not limited to, diabetes (e.g., type 1 diabetes,Type 2 diabetes, or gestational diabetes), non-alcoholic fatty liverdisease (NAFLD), non-alcoholic steatohepatitis (NASH), and/or obesity.

The present invention provides an antibody peptide conjugate comprisingan antibody having a light chain having the amino acid sequence of SEQID NO:64 and a heavy chain having the amino acid sequence of SEQ IDNO:65 conjugated to a peptide having the amino acid sequence

(SEQ ID NO: 62) His-Xaa²-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Xaa¹⁶-Arg-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asp-Thr-Lys-Gln

wherein Xaa² is α-aminoisobutyric acid (aib) or D-Ser; Xaa¹⁶ is aib; atleast one of the amino acids at position 10, 24, or 31 is substitutedwith a Lys or the amino acids at positions 10 and 24 are eachsubstituted with a Lys; and optionally the peptide comprises up to threeadditional amino acid substitutions; wherein the heavy chain includes asubstitution of the amino acid a position 16, 32, 33, 56, 114, 125, 142,179, 198, or 211 with a para-acetylphenylalanine (pAcF) or the lightchain includes a substitution of the amino acid a position 125 or 142with a pAcF; wherein the peptide comprises either (i) an aminoxy acidresidue covalently linked to the epsilon amino a group of the Lys atposition 10, 24, or 31 or (ii) an aminoxy acid residue covalently linkedto the epsilon amino a group of the Lys at position 10, 24, or 31 via apolyethylene glycol (PEG) spacer, a γGlu spacer, or a γGlu-γGlu spacer;wherein the pAcF residue is covalently linked to the aminooxy acidresidue; and wherein the antibody peptide conjugate is an agonist of theglucagon receptor and the glucagon-like peptide 1 receptor. Inparticular aspects of the antibody peptide conjugate, the peptidecomprises a fatty acid acid covalently linked to the epsilon amino groupof the Lys at position 10 via a γGlu-γGlu spacer. In particular aspectsof the antibody peptide conjugate, the fatty acid comprises a C14, C15,C16, C17, C18, C19, or C20 fatty acid. In particular aspects of theantibody peptide conjugate, the fatty acid comprises a C14 fatty acid.

In particular aspects of the antibody peptide conjugate, thepolyethylene glycol spacer comprises 2, 4, 6, 8, 24, or 36 ethoxy units.

The present invention further provides an antibody peptide conjugatecomprising an antibody having a light chain having the amino acidsequence of SEQ ID NO:64 and a heavy chain having the amino acidsequence of SEQ ID NO:65 conjugated to a peptide having the amino acidsequence

(SEQ ID NO: 63) His-Xaa²-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa¹⁰-Ser-Xaa¹²-Tyr-Leu-Asp-Xaa¹⁶-Arg-Ala-Ala-Xaa²⁰-Asp-Phe-Val-Xaa²⁴-Xaa²⁵-Xaa²⁶-Xaa²⁷-Xaa²⁸-Xaa²⁹-Lys-Xaa³¹Wherein Xaa² is α-aminoisobutyric acid (aib) or D-Ser; Xaa¹⁶ is aib;Xaa³¹ is Lys or Gln when Xaa¹⁰ or Xaa²⁴ is Lys with the proviso that forany peptide only one of Xaa¹⁰, Xaa²⁴, or Xaa³¹ can be Lys; Xaa¹⁰ is Tyrwhen Xaa³¹ is Lys, or Lys if neither Xaa²⁴ or Xaa³¹ is Lys, or Tyr orLys if Xaa²⁴ is Lys; X¹² is Lys or aib when Xaa³¹ is Lys, with theproviso that when Xaa¹² is aib, then either Xaa²⁰ or Xaa²⁴ is aib andXaa¹⁰, Xaa²⁵, Xaa²⁶, Xaa²⁵, Xaa²⁷, Xaa²⁸, and Xaa²⁹ are Tyr, Trp, Leu,Met, Asp, and Thr, respectively; Xaa²⁰ is Gln or aib when Xaa²⁴ or Xaa³¹is Lys with the proviso that when Xaa²⁰ is aib then Xaa¹⁰, Xaa²⁴, Xaa²⁵,Xaa²⁶, Xaa²⁵, Xaa²⁷, Xaa²⁸, and Xaa²⁹ are Tyr, Gln, Trp, Leu, Met, Asp,and Thr, respectively; Xaa²⁴ is Gln when Xaa¹⁰ is Lys, or Gln or aibwhen Xaa³¹ is Lys, or Lys when Xaa¹⁰ and Xaa³¹ are Tyr and Gln,respectively, or Glu when Xaa²⁸ is Lys, with the proviso that when Xaa²⁴is aib then Xaa¹⁰, Xaa²⁵, Xaa²⁶, Xaa²⁷, Xaa²⁸, an Xaa²⁹ are Tyr, Trp,Leu, Met, Asp, and Thr, respectively; Xaa²⁵ is Trp, Gln, Asp, Lys, aib,alpha-methylphenylanine, (αMePhe), alpha-methyl-leucine (αMeLeu),alpha-methyltryptophan (αMeTrp), beta-homo tryptophan (ßhoTrp),5-hydroxy tryptophane (W(5OH)), ß-3-benzothienyl)-alanine (Bzt),7-azatryptophan (AzTrp), 2-naphthylalanine (2Nap), (3-Pyridyl)alanine(3Pyr), 3-(2-quinoyl)-alanine (3Qui), 4,4′-biphenylalanine (BIP), orN-methyl-tryptophane (Trp(Me)) with the proviso that when Xaa²⁵ is Gln,Asp, Lys, aib, αMePhe, αMeLeu, αMeTrp, ßhoTrp, W(5OH), Bzt, AzTrp, 2Nap,3Pyr, 3-(2-quinoyl)-alanine (3Qui), 4,4′-biphenylalanine (BIP), orN-methyl-tryptophane (Trp(Me)), then Xaa¹², Xaa²⁰, Xaa²⁴, Xaa²⁶, andXaa²⁷ are Lys, Gln, Gln, Leu, and Met, respectively, and Xaa³¹ is Lys;Xaa²⁶ is Leu, aib, or hexafluoroleucine (HFL) with the proviso that whenXaa²⁶ is aib or HFL, then Xaa³¹ is Lys and Xaa¹², Xaa²⁰, Xaa²⁴, Xaa²⁵,and Xaa²⁷ are Lys, Gln, Gln, Trp, and Met, respectively; Xaa²⁷ is Met,methionine sulfoxide (M(O)), homoleucine (hLeu), or homocycloexylalanine(hCha) with the proviso that when Xaa²⁷ is M(O), hLeu, or hCha, thenXaa³¹ is Lys and Xaa¹², Xaa²⁰, Xaa²⁴, Xaa²⁵, Xaa²⁶, Xaa²⁸, Xaa²⁹ areLys, Gln, Gln, Trp, Leu, Asp, and Thr, respectively; Xaa²⁸ is Asp oraib, or Lys when Xaa²⁴ is Glu, with the proviso that when Xaa²⁸ includesaib then Xaa²⁴ is Lys and Xaa¹⁰, Xaa¹², Xaa²⁵, Xaa²⁶, Xaa²⁷, Xaa²⁹ areTyr, Lys, Trp, Leu, Met, and Thr, respectively; Xaa²⁹ is Thr or aib withthe proviso that when Xaa²⁹ is aib then Xaa³¹ is Lys and Xaa¹⁰, Xaa¹²,Xaa²⁵, Xaa²⁶, Xaa²⁷, Xaa²⁸ are Tyr, Lys, Trp, Leu, Met, Asp, and Thr,respectively; one of Lys¹⁰, Lys²⁴, or Lys³¹ is covalently linked at itsepsilon amino group to an aminooxy acid residue; optionally, Lys¹⁰ maybe covalently linked at its epsilon amino group to a fatty acid if Lys²⁴is covalently linked to aminooxy acid residue; wherein either the lightchain includes a substitution of the amino acid at position 125 or 142with a para-acetylphenylalanine (pAcF) or the heavy chain includes asubstitution of the amino acid a position 16, 32, 33, 56, 114, 179, 198,or 211 with a AcF; and wherein the aminooxy acid residue of the peptideis covalently linked to the pAcF residue of the antibody.

In particular aspects of the antibody peptide conjugate, the peptidecomprises the Lys¹⁰, Lys²⁴, or Lys³¹ covalently linked at the epsilonamino group directly to the aminooxy acid residue or to the aminooxyacid residue via a polyethylene glycol spacer, γGlu spacer, or γGlu-γGluspacer.

In particular aspects of the antibody peptide conjugate, thepolyethylene glycol spacer comprises 2, 4, 6, 8, 24, or 35 ethoxy units.In particular aspects of the antibody peptide conjugate, the peptidecomprises the Lys¹⁰ covalently linked to a fatty acid acid via aγGlu-γGlu spacer. In particular aspects of the antibody peptideconjugate, the fatty acid comprises a C14, C15, C16, C17, C18, C19, orC20 fatty acid. In particular aspects of the antibody peptide conjugate,the fatty acid comprises a C14 fatty acid.

In particular aspects of the antibody peptide conjugate, the peptidecomprises Lys³¹ covalently linked at the epsilon amino group directly tothe aminooxy acid residue or to the aminooxy acid residue via apolyethylene glycol spacer, γGlu spacer, or γGlu-γGlu spacer.

In particular aspects of the antibody peptide conjugate, the peptidecomprises a Glu at Xaa²⁴ and a Lys at Xaa²⁸ and a lactam bridge betweenthe Glu and the Lys.

The present invention further provides an antibody peptide conjugatecomprising:

an antibody having a light chain having the amino acid sequence of SEQID NO:64, SEQ ID NO:71, or SEQ ID NO:72 and a heavy chain having theamino acid sequence of selected from SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:73, SEQ IDNO:74, or SEQ ID NO:75 conjugated to a peptide comprising the amino acidsequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52,SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57,SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, or SEQ ID NO:61, with theproviso that if the light chain has the amino acid sequence of SEQ IDNO:64 then heavy chain does not have the amino acid sequence of SEQ IDNO:65 and if the light chain has the amino acid sequence of SEQ ID NO:71or 72 then the heavy chain has the amino acid sequence of SEQ ID NO:65.

The present invention further provides an antibody peptide conjugatecomprising an antibody having a light chain having the amino acidsequence of SEQ ID NO:64 and a heavy chain having the amino acidsequence of SEQ ID NO:66 conjugated to a peptide have the amino acidsequence of SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:58, or SEQ ID NO:60.

The present invention further provides a pharmaceutical compositioncomprising the antibody peptide conjugate of any one of the previousclaims and a pharmaceutically acceptable carrier.

The present invention further provides a kit comprising thepharmaceutical composition and a device for administering thepharmaceutical composition to a patient, optionally, wherein the devicecomprises a syringe comprising the pharmaceutical composition.

The present invention further provides a method for treating a metabolicdisease or disorder in a patient, comprising administering to a patientin need the pharmaceutical composition to treat the metabolic disease ordisorder.

The present invention further provides the pharmaceutical compositionfor use in the treatment of a metabolic disease or disorder.

The present invention further provides the use of the pharmaceuticalcomposition for the manufacture of a medicament to treat the metabolicdisease or disorder.

In particular aspects, the metabolic disease or disorder comprisesdiabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholicsteatohepatitis (NASH), or obesity. In particular aspects the diabetescomprises Type 1, diabetes, Type II diabetes, or gestational diabetes.

The present invention further provides an antibody comprising a lightchain having the amino acid sequence of SEQ ID NO:64, SEQ ID NO:71, orSEQ ID NO:72 and a heavy chain having the amino acid sequence ofselected from SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68,SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75with the proviso that if the light chain has the amino acid sequence ofSEQ ID NO:64 then heavy chain does not have the amino acid sequence ofSEQ ID NO:65 and if the light chain has the amino acid sequence of SEQID NO:71 or 72 then the heavy chain has the amino acid sequence of SEQID NO:65.

The present invention further provides an antibody comprising antibodycomprising a light chain having the amino acid sequence of SEQ ID NO:64and a heavy chain having the amino acid sequence of SEQ ID NO:65;wherein the heavy chain includes a substitution of the amino acid aposition 16, 32, 33, 56, 114, 179, 198, or 211 with apara-acetylphenylalanine (pAcF).

The present invention further provides for the use of the antibody forthe manufacture of a medicament for the treatment of a metabolic diseaseor disorder.

In particular aspects, the metabolic disease or disorder comprisesdiabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholicsteatohepatitis (NASH), or obesity. In particular aspects the diabetescomprises Type 1, diabetes, Type II diabetes, or gestational diabetes.

Definitions

The term “about” as used herein means greater or lesser than the valueor range of values stated by 10 percent, but is not intended todesignate any value or range of values to only this broader definition.Each value or range of values preceded by the term “about” is alsointended to encompass the embodiment of the stated absolute value orrange of values.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of theU.S. Federal government or listed in the U.S. Pharmacopeia for use inanimals, including humans.

As used herein the term “pharmaceutically acceptable salt” refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto.

Pharmaceutically acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms. For example, as used herein the term “treating diabetes” willrefer in general to altering glucose blood levels in the direction ofnormal levels and may include increasing or decreasing blood glucoselevels depending on a given situation.

As used herein an “effective” amount or a “therapeutically effectiveamount” of a glucagon peptide refers to a nontoxic but sufficient amountof the peptide to provide the desired effect. For example one desiredeffect would be the prevention or treatment of hyperglycemia, e.g., asmeasured by a change in blood glucose level closer to normal, orinducing weight loss/preventing weight gain, e.g., as measured byreduction in body weight, or preventing or reducing an increase in bodyweight, or normalizing body fat distribution. The amount that is“effective” will vary from subject to subject, depending on the age andgeneral condition of the individual, mode of administration, and thelike. Thus, it is not always possible to specify an exact “effectiveamount.” However, an appropriate “effective” amount in any individualcase may be determined by one of ordinary skill in the art using routineexperimentation.

The term, “parenteral” means not through the alimentary canal but bysome other route, e.g., subcutaneous, intramuscular, intraspinal, orintravenous.

As used herein, the term “peptide” encompasses a chain of 3 or moreamino acids and typically less than 100 amino acids, wherein the aminoacids are naturally occurring or coded or non-naturally occurring ornon-coded amino acids. Non-naturally occurring amino acids refer toamino acids that do not naturally occur in vivo but which, nevertheless,can be incorporated into the peptide structures described herein.“Non-coded” as used herein refers to an amino acid that is not anL-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu,Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val,Trp, Tyr. “Coded” as used herein refers to an amino acid that is anL-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu,Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val,Trp, Tyr. In some embodiments, the peptides and variant peptidesdescribed herein are about the same length as SEQ ID NO: 1 (which is 29amino acids in length), e.g. 25-35 amino acids in length. Exemplarylengths include 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.

Typically, polypeptides and proteins have a polymer length that isgreater than that of “peptides.”

Amino acid “modification” refers to an insertion, deletion orsubstitution of one amino acid with another. In some embodiments, theamino acid substitution or replacement is a conservative amino acidsubstitution, e.g., a conservative substitution of the amino acid at oneor more of positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20,21, 24, 27, 28 or 29. As used herein, the term “conservative amino acidsubstitution” is the replacement of one amino acid with another aminoacid having similar properties, e.g., size, charge, hydrophobicity,hydrophilicity, and/or aromaticity, and includes exchanges within one ofthe following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

Ala, Ser, Thr, Pro, Gly;

II. Polar, negative-charged residues and their amides and esters:

Asp, Asn, Glu, Gin, cysteic acid and homocysteic acid;

III. Polar, positive-charged residues:

His, Arg, Lys; Ornithine (Orn)

IV. Large, aliphatic, nonpolar residues:

Met, Leu, He, Val, Cys, Norleucine (Nle), homocysteine

V. Large, aromatic residues:

Phe, Tyr, Trp, acetyl phenylalanine

In some embodiments, the amino acid substitution is not a conservativeamino acid substitution, e.g., is a non-conservative amino acidsubstitution.

As used herein the term “charged amino acid” or “charged residue” refersto an amino acid that comprises a side chain that is negative-charged(i.e., de-protonated) or positive-charged (i.e., protonated) in aqueoussolution at physiological pH. For example negative-charged amino acidsinclude aspartic acid, glutamic acid, cysteic acid, homocysteic acid,and homoglutamic acid, whereas positive-charged amino acids includearginine, lysine and histidine. Charged amino acids include the chargedamino acids among the 20 coded amino acids, as well as atypical ornon-naturally occurring or non-coded amino acids.

As used herein the term “acidic amino acid” refers to an amino acid thatcomprises a second acidic moiety (other than the carboxylic acid of theamino acid), including for example, a carboxylic acid or sulfonic acidgroup.

As used herein, the term “acylated amino acid” refers to an amino acidcomprising an acyl group which is non-native to a naturally-occurringamino acid, regardless of the means by which it is produced (e.g.acylation prior to incorporating the amino acid into a peptide, oracylation after incorporation into a peptide).

As used herein the term “alkylated amino acid” refers to an amino acidcomprising an alkyl group which is non-native to a naturally-occurringamino acid, regardless of the means by which it is produced.Accordingly, the acylated amino acids and alkylated amino acids of thepresent disclosures are non-coded amino acids.

As used herein, the term “selectivity” of a molecule for a firstreceptor relative to a second receptor refers to the following ratio:EC₅₀ of the molecule at the second receptor divided by the EC₅₀ of themolecule at the first receptor. For example, a molecule that has an EC50of 1 nM at a first receptor and an EC₅₀ of 100 nM at a second receptorhas 100-fold selectivity for the first receptor relative to the secondreceptor.

As used herein, “glucagon potency” or “potency compared to nativeglucagon” of a molecule refers to the inverse ratio of the EC₅₀ of themolecule at the glucagon receptor divided by the EC₅₀ of native glucagonat glucagon receptor.

As used herein, “GLP-1 potency” or “potency compared to native GLP-1” ofa molecule refers to the inverse ratio of the EC₅₀ of the molecule atGLP-1 receptor divided by the EC₅₀ of native GLP-1 at GLP-1 receptor.

As used herein, the terms “antibody,” “immunoglobulin,”“immunoglobulins” and “immunoglobulin molecule” are usedinterchangeably. Each immunoglobulin molecule has a unique structurethat allows it to bind its specific antigen, but all immunoglobulinshave the same overall structure as described herein. The basicimmunoglobulin structural unit is known to comprise a tetramer ofsubunits. Each tetramer has two identical pairs of polypeptide chains,each pair having one “light” chain (about 25 kDa) and one “heavy” chain(about 50-70 kDa). The amino-terminal portion of each chain includes avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The carboxy-terminal portion ofeach chain defines a constant region primarily responsible for effectorfunction. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, and definethe antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively.

The light and heavy chains are subdivided into variable regions andconstant regions (See generally, Fundamental Immunology (Paul, W., ed.,2nd ed. Raven Press, N.Y., 1989), Ch. 7. The variable regions of eachlight/heavy chain pair form the antibody binding site. Thus, an intactantibody has two binding sites. Except in bifunctional or bispecificantibodies, the two binding sites are the same. The chains all exhibitthe same general structure of relatively conserved framework regions(FR) joined by three hypervariable regions, also called complementaritydetermining regions or CDRs. The CDRs from the two chains of each pairare aligned by the framework regions, enabling binding to a specificepitope. The terms include naturally occurring forms, as well asfragments and derivatives. Included within the scope of the term areclasses of immunoglobulins (Igs), namely, IgG, IgA, IgE, IgM, and IgD.Also included within the scope of the terms are the subtypes of IgGs,namely, IgG1, IgG2, IgG3, and IgG4. The term is used in the broadestsense and includes single monoclonal antibodies (including agonist andantagonist antibodies) as well as antibody compositions which will bindto multiple epitopes or antigens.

The term “monoclonal antibody” (mAb) as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. The term “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., (1975) Nature, 256:495, or may be made byrecombinant DNA methods (See, for example, U.S. Pat. No. 4,816,567; thedisclosure of which is incorporated herein by reference).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the general structure of an antibody peptideconjugate and its synthesis. The antibody is shown with twopara-acetylphenylalanine residues conjugated to the heavy chain of theantibody. A peptide comprising an aminooxy functional reactive groupconjugated to the lysine residue at position 31 of the peptide via apolyethylene spacer is conjugated to the antibody under conditionsappropriate for the NH₂ of the aminooxy group to react with the sideacetyl group of the paracetylphenylalanine residue in the antibody aminoacid sequence to produce an antibody with two peptides conjugatedthereto.

FIG. 2 shows a graph of the cumulative weight change (grams) ofdiet-induced obese (DIO) mice treated with Ambody-P4740 @ 3 mg/kg,Ambody-P4704 @ 3 and 10 mg/kg, Ambody-P5615 @ 3 and 10 mg/kg, singledose via tail vein injection on day 0.

FIG. 3 shows a graph of the cumulative body weight change (%) relativeto Ambody-P4740, a control group of DIO mice treated with a single IVdose of an Ambody-P4704, Ambody-P5615.

FIG. 4 shows a graph of the cumulative change in food intake of DIO micetreated with Ambody-P4740 @ 3 mg/kg, Ambody-P4704 @ 3 and 10 mg/kg,Ambody-P5615 @ 3 and 10 mg/kg, single dose via tail vein at day 0.

FIG. 5 shows the plasma concentration in DIO mice treated withAmbody-4740 @ 3 mg/kg, Ambody-4704 @ 3 and 10 mg/kg, Ambody-5615 @ 3 and10 mg/kg, single dose via tail vein at day 1, 3, 7 and 10. Values aremean±SD, n=4.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosures provide antibody peptide conjugates (APCs) inwhich an antibody is conjugated to peptides that exhibit activity at theGLP-1 receptor (GLP-1) and the glucagon (GCG) receptor. In this regard,the present disclosures provide APCs conjugated to GLP-1 receptor/GCGreceptor co-agonist peptides. In exemplary embodiments, the presentlydisclosed APCs exhibit activity or potency at the GCG receptor and/orthe GLP-1 receptors.

In exemplary embodiments, the APCs described herein exhibit otherimprovements in properties relative to native glucagon or native GLP-1,such as greater stability, greater solubility, a prolonged half-life incirculation, a delayed onset of action, an extended duration of action,a dampened peak (e.g., relatively decreased mean peak plasmaconcentration), and an improved resistance to proteases, such as DPP-IV.

Glucagon is a peptide hormone structurally related to GLP-1 that is wellrecognized for its acute ability to increase blood glucose throughstimulation of glycogenolysis and gluconeogenesis. While administrationof glucagon was first reported over 60 years ago to yield improvementsin rodent metabolism, including lowering body weight (Salter, Am. J.Clin. Nutr. 8: 535-539 (1960)) these results have not been translatedinto the use of glucagon in therapies for a treatment of obesity inhumans, particularly due to the inherent risk of hyperglycemia,especially in insulin-resistant type-2 diabetic patients.

The use of GCG receptor/GLP-1 receptor co-agonists as being of enhancedefficacy and safety relative to pure GLP-1 receptor agonists in thetreatment of rodent obesity, with simultaneous improvement in glycemiccontrol was disclosed by Day et al. in Nat. Chem. Biol. 5: 749-757(2009) and Pocai et al. in Diabetes 58: 2258-2266 (2009). Oxyntomodulin(OXM) is an endogenous precursor to glucagon, which is secretedpostprandially by L-cells of the jejuno-ileum together with GLP-1 andhas been shown to be a balanced co-agonist at the GLP-1 receptor andglucagon receptor albeit of relatively low potency (Hoist, Regul. Pept.93: 45-51 (2000); Drucker, J. Nat. Clin. Pract. Endocrinol. Metab. 1:22-31 (2005); Baldissera et al., Regul. Pept. 21: 151-166 (1988); Groset al., Endocrinol. 133: 631-638 (1993); Pocai et al., op. cit.). A4-week clinical study in obese subjects demonstrated that repeatedsubcutaneous administration of OXM was well tolerated and causedsignificant weight loss, with a concomitant reduction in food intake(Wynne et al., Diabetes 54: 2390-2395 (2005)).

Antibodies, such as IgG are “Y”-shaped macromolecules called monomers. Amonomer is composed of four glycoprotein chains: two identical heavychains and two identical light chains. The two heavy chains have a highmolecular weight that varies with the class of antibody. The lightchains come in two varieties: kappa or lambda and have a lower molecularweight than the heavy chains. The four glycoprotein chains are connectedto one another by disulfide (S—S) bonds and non-covalent bonds (see FIG.1). Additional S—S bonds fold the individual glycoprotein chains into anumber of distinct globular domains. The area where the top of the “Y”joins the bottom is called the hinge. This area is flexible to enablethe antibody to bind to pairs of epitopes various distances apart on anantigen.

The APCs of the present invention comprises the co-agonist peptideconjugated to an Ambody, which is an antibody derived from Palivizumabthat comprises a pAcF residue in the heavy chains. Because each APC iscomprised of two identical light chains and two identical heavy chains,each APC of the present invention comprises at least two co-agonistpeptides (See FIG. 1).

Table 1 shows exemplary GCG receptor/GLP-1 receptor co-agonist peptideswhich may be conjugated to the pAcF of an Ambody as disclosed herein toprovide an APC as disclosed herein.

TABLE 1 SEQ ID NO: # Peptide Structure  1 4704HXQGTFTSDYSKYLDXRAAQDFVQWLMDTK-K(COCH₂ONH₂)-CONH₂  2 4739HXQGTFTSDYSKYLDXRAAQDFVQWLMDTK-K(PEG2-COCH₂ONH₂)-CONH₂  3 4740HXQGTFTSDYSKYLDXRAAQDFVQWLMDTK-K(PEG4-COCH₂ONH₂)-CONH₂  4 5058HXQGTFTSDYSKYLDXRAAQDFVQWLMDTK-K(PEG6-COCH₂ONH₂)-CONH₂  5 5059HXQGTFTSDYSKYLDXRAAQDFVQWLMDTK-K(PEG8-COCH₂ONH₂)-CONH₂  6 5615HXQGTFTSDYSKYLDXRAAQDFVQWLMDTK-K(PEG24-COCH₂ONH₂)-CONH₂  7 6115HXQGTFTSDYSKYLDXRAAQDFVQWLMDTK-K(PEG36-COCH₂ONH₂)-CONH₂  8 5275HXDGTFTSDYSKYLDXRAAQDFVQWLMDTK-K(PEG4-COCH₂ONH₂)-CONH₂  9 5048HsQGTFTSDYSKYLDXRAAQDFVQWLMDTK-K(PEG2-COCH₂ONH₂)-CONH₂ 10 5049HsQGTFTSDYSKYLDXRAAQDFVQWLMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 11 5050HsQGTFTSDYSKYLDXRAAQDFVQWLMDTK-K(PEG6-COCH₂ONH₂)-CONH₂ 12 5051HsQGTFTSDYSKYLDXRAAQDFVQWLMDTK-K(PEG8-COCH₂ONH₂)-CONH₂ 13 6114HsQGTFTSDYSKYLDXRAAQDFVQWLMDTK-K(PEG24-COCH₂ONH₂)-CONH₂ 14 4840HXQGTFTSDYSKYLDXRAAQDFV-K(COCH₂ONH₂)-WLMDTKQ-COOH 15 4841HXQGTFTSDYSKYLDXRAAQDFV-K(PEG2-COCH₂ONH₂)-WLMDTKQ-COOH 16 4842HXQGTFTSDYSKYLDXRAAQDFV-K(PEG4-COCH₂ONH₂)-WLMDTKQ-COOH 17 5009HXQGTFTSDYSKYLDXRAAQDFV-K(PEG6-COCH₂ONH₂)-WLMDTKQ-COOH 18 5010HXQGTFTSDYSKYLDXRAAQDFV-K(PEG8-COCH₂ONH₂)-WLMDTKQ-COOH 19 5799HXQGTFTSDYSKYLDXRAAXDFV-K(PEG4-COCH₂ONH₂)-WLMXTKQ-COOH 20 5052HsQGTFTSDYSKYLDXRAAQDFV-K(PEG2-COCH₂ONH₂)-WLMDTKQ-COOH 21 5053HsQGTFTSDYSKYLDXRAAQDFV-K(PEG4-COCH₂ONH₂)-WLMDTKQ-COOH 22 5314HsQGTFTSDYSKYLDXRAAQDFV-K(PEG2-COCH₂ONH₂)-WLMDT-CONH₂ 23 5420HsQGTFTSDYSKYLDXRAAQDFV-K(PEG4-COCH₂ONH₂)-WLMDT-CONH₂ 24 5798HXQGTFTSDYSKYLDXRAAQDFVEWLMKTK-K(PEG4-COCH₂ONH₂)-CONH₂(lactam bridge between E and K) 25 5759HXQGTFTSDYSKYLDXRAAQDFVQ-Bzt-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 26 5760HXQGTFTSDYSKYLDXRAAQDFVQ-AzTrp-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 27 5761HXQGTFTSDYSKYLDXRAAQDFVQ-2Nap-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 28 5762HXQGTFTSDYSKYLDXRAAQDFVQ-3Pyr-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 29 5763HXQGTFTSDYSKYLDXRAAQDFVQWLMDXK-K(PEG4-COCH₂ONH₂)-CONH₂ 30 5764HXQGTFTSDYSKYLDXRAAQDFVQWL-M(O)-DTK-K(PEG4-COCH₂ONH₂)-CONH₂ 31 5765HXQGTFTSDYSKYLDXRAAQDFVQWL-hLeu-DTK-K(PEG4-COCH₂ONH₂)-CONH₂ 32 5766HXQGTFTSDYSKYLDXRAAQDFVQWL-hCha-DTK-K(PEG4-COCH₂ONH₂)-CONH₂ 33 6052HXQGTFTSDYSKYLDXRAAQDFVQ-3Qui-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 34 6053HXQGTFTSDYSKYLDXRAAQDFVQ-Bip-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 35 6062HXQGTFTSDYSKYLDXRAAQDFVQ-Trp(Me)-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 36 6376HXQGTFTSDYSKYLDXRAAQDFVQWXMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 37 6377HXQGTFTSDYSKYLDXRAAQDFVQW-HFL-MDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 38 6378HXQGTFTSDYSKYLDXRAAQDFVQ-W(5OH)-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 39 6379HXQGTFTSDYSKYLDXRAAQDFVQ-Lys-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 40 6380HXQGTFTSDYSKYLDXRAAQDFVQ-Gln-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 41 6381HXQGTFTSDYSKYLDXRAAQDFVQ-Asn-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 42 6382HXQGTFTSDYSKYLDXRAAQDFVQ-αMePhe-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 43 6383HXQGTFTSDYSKYLDXRAAQDFVQXLMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 44 6384HXQGTFTSDYSKYLDXRAAQDFVQ-αMeLeu-LMDTK-K(PEG4-COCH₂ONH₂-CONH2 45 6385HXQGTFTSDYSKYLDXRAAQDFVQ-αMeTrp-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 46 6386HXQGTFTSDYSKYLDXRAAQDFVQ-βhoTrp-LMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 47 4699HXQGTFTSD-K(COCH₂ONH₂)-SKYLDXRAAQDFVQWLMDTKQ-COOH 48 4700HXQGTFTSD-K(γE-COCH₂ONH₂)-SKYLDXRAAQDFVQWLMDTKQ-COOH 49 4701HXQGTFTSD-K(γEγE-COCH₂ONH₂)-SKYLDXRAAQDFVQWLMDTKQ-COOH 50 4702HXQGTFTSD-K(Peg2-COCH₂ONH₂)-SKYLDXRAAQDFVQWLMDTKQ-COOH 51 4703HXQGTFTSD-K(Peg4-COCH₂ONH₂)-SKYLDXRAAQDFVQWLMDTKQ-COOH 52 4896HXQGTFTSD-K(γEγE-Palm)SKYLDXRAAQDFV-K(COCH₂ONH₂)-WLMDTKQ-COOH 53 4952HXQGTFTSD-K(γEγE-Palm)-SKYLDXRAAQDFV-K(PEG2-COCH₂ONH₂)-WLMDTKQ-COOH 544953 HXQGTFTSD-K(γEγE-Palm)-SKYLDXRAAQDFV-K(PEG4-COCH₂ONH₂)-WLMDTKQ-COOH55 5866 HSQGTFTSDYSKYLSDRAAQDFVQWLMDTK-K(PEG4-COCH₂ONH₂)-CONH₂ 56 6541HXQGTFTSDYSKYLDXRAAQDFVXWLMXTK-K(PEG24-COCH₂ONH₂)-CONH₂ 57 6542HXQGTFTSDYSXYLDXRAAQDFVXWLMXTK-K(PEG24-COCH₂ONH₂)-CONH₂ 58 6543HXQGTFTSDYSKYLDXRAAXDFVQWLMXTK-K(PEG24-COCH₂ONH₂)-CONH₂ 59 6544HXQGTFTSDYSXYLDXRAACDFVQWLMXTK-(PEG24-COCH₂ONH₂)-CONH₂ 60 6545HXQGTFTSDYSKYLDXRAAXDFVK(PEG24-COCH₂ONH₂)-WLMXTKQ-COOH 61 6546HXQGTFTSDYSXYLDXRAAXDFVK(PEG24-COCH₂ONH₂)-WLMXTKQ-COOH X =α-aminobutyric acid (Aib); s = D-Ser; β-(3-benzothienyl)-alanine; AzTrp= 7 Azatryptophan; 2Nap = 2-naphthylalanine; 3Pyr = (3-Pyridyl)alanine;M(O) = methionine sulfoxide; hLeu = homoleucine; hCha =homocycloexylalanine; 3Qui = 3-(2quinoyl)-alanine; Bip =4,4'-biphenylalanine; Trp(Me) = N-methyl-tryptophane; HFL =hexafluoroleucine; W(5OH) = 5-hydroxy tryptophan; αMePhe =alpha-methylphenylalanine; αMeLeu = alpha-methyl-leucine; αMeTrp =alpha-methyltryptophan; βhoTrp = beta homo tryptophan; γE = gammaglutamic acid; PEG = polyethylene glycol, wherein the interger indicatesthe number of ethylene units; Palm = palmitoyl (C15-CO-)

The Ambody is a derivative of palivizumab in which an amino acid atposition 16, 32, 33, 56, 114, 179, 198, or 211 of the heavy chain isreplaced with the non-natural amino acid para-acetylphenylalanine (pAcF)or an amino acid at position 125 or 142 of the light chain is replacedwith pAcF. In particular embodiments, the light chain has the amino acidsequence shown in SEQ ID NO:64, SEQ ID NO:71, or SEQ ID NO:72 and theheavy chain has the amino acid shown in SEQ ID NO:65 or the amino acidsequence in SEQ ID NO:65 wherein the amino acid at position 16, 32, 33,56, 114, 179, 198, or 211 has been replaced with pAcF with the provisothat if the light chain has the amino acid sequence of SEQ ID NO:71 orSEQ ID NO:72 then the heavy chain has the amino acid sequence shown inSEQ ID NO:65, or if the light chain has the amino acid sequence of SEQID NO:64 then the heavy chain has the amino acid sequence of SEQ IDNO:65 wherein at least one of positions 16, 32, 33, 56, 114, 179, 198,or 211 has been replaced with the non-natural amino acid pAcF. Aminoacid sequences for each of the aforementioned heavy chains having asubstitution of pAcF for the amino acid at position 16, 32, 33, 56, 114,179, 198, or 211 are shown by SEQ ID NO:66, 67, 68, 69, 70, 73, 74, or75, respectively.

Para-acetylphenylalanine has the structure

In a particular aspect, the pAcF residue of the ambody may be conjugatedto the peptide via the aminooxy residue, which is directly conjugated tothe epsilon amino group of a Lys residue of the peptide, in a linkagehaving the structure

wherein the bond between the epsilon amino group of the Lys residue andthe aminooxy residue is indicated by wavy line a and the bond betweenamino group of the aminooxy residue and the pAcF is indicated by wavyline b.

In a particular aspect, the pAcF residue of the ambody may be conjugatedto the peptide via aminooxy residue, which is conjugated to the epsilonamino group of a Lys residue of the peptide via an amino polyethyleneglycol spacer, in a linkage having the structure

wherein the bond between the epsilon amino group of the Lys residue andthe amino polyethylene gycol spacer is indicated by wavy line c and thebond between amino group of the aminooxy residue and the pAcF isindicated by wavy line d and wherein n is 1, 3, 5, 7, 23, or 35.

In a particular aspect, the pAcF residue of the ambody may be conjugatedto the peptide via aminooxy residue, which is conjugated to the epsilonamino group of a Lys residue of the peptide via an amino polyethyleneglycol spacer (Peg2), in a linkage having the structure

wherein the bond between the epsilon amino group of the Lys residue andthe amino polyethylene gycol spacer is indicated by wavy line c and thebond between amino group of the aminooxy residue and the pAcF isindicated by wavy line d.

In a particular aspect, the pAcF residue of the ambody may be conjugatedto the peptide via aminooxy residue, which is conjugated to the epsilonamino group of a Lys residue of the peptide via an amino polyethyleneglycol spacer (Peg4), in a linkage having the structure

wherein the bond between the epsilon amino group of the Lys residue andthe amino polyethylene gycol spacer is indicated by wavy line c and thebond between amino group of the aminooxy residue and the pAcF isindicated by wavy line d.

In a particular aspect, the pAcF residue of the ambody may be conjugatedto the peptide via aminooxy residue, which is conjugated to the epsilonamino group of a Lys residue of the peptide via an amino polyethyleneglycol spacer (Peg8), in a linkage having the structure

wherein the bond between the epsilon amino group of the Lys residue andthe amino polyethylene gycol spacer is indicated by wavy line c and thebond between amino group of the aminooxy residue and the pAcF isindicated by wavy line d.

In a particular aspect, the pAcF residue of the ambody may be conjugatedto the peptide via aminooxy residue, which is conjugated to the epsilonamino group of a Lys residue of the peptide via an amino polyethyleneglycol spacer (Peg24), in a linkage having the structure

wherein the bond between the epsilon amino group of the Lys residue andthe amino polyethylene gycol spacer is indicated by wavy line c and thebond between amino group of the aminooxy residue and the pAcF isindicated by wavy line d.

In a particular aspect, the pAcF residue of the ambody may be conjugatedto the peptide via aminooxy residue, which is conjugated to the epsilonamino group of a Lys residue of the peptide via an amino polyethyleneglycol spacer (Peg36), in a linkage having the structure

wherein the bond between the epsilon amino group of the Lys residue andthe amino polyethylene gycol spacer is indicated by wavy line c and thebond between amino group of the aminooxy residue and the pAcF isindicated by wavy line d.

In a particular aspect, the pAcF residue of the ambody may be conjugatedto the peptide via aminooxy residue, which is conjugated to the epsilonamino group of a Lys residue of the peptide via a gamma-Glu (γE) spacer,in a linkage having the structure

wherein the bond between the epsilon amino group of the Lys residue andthe γE spacer is indicated by wavy line e and the bond between aminogroup of the aminooxy residue and the pAcF is indicated by wavy line fand wherein n is 1, 3, 5, 7, 23, or 37.

In a particular aspect, the pAcF residue of the ambody may be conjugatedto the peptide via aminooxy residue, which is conjugated to the epsilonamino group of a Lys residue of the peptide via a gamma-Glu-gamma-Glu(γEyE) spacer, in a linkage having the structure

wherein the bond between the epsilon amino group of the Lys residue andthe γEyE spacer is indicated by wavy line e and the bond between aminogroup of the aminooxy residue and the pAcF is indicated by wavy line fand wherein n is 1, 3, 5, 7, 23, or 37.

The ambodies may be modified to include a pAcF residue using theorthoganol tRNA technology disclosed in for example U.S. Pat. Nos.7,045,337; 7,632,924; and 7,723,070, each incorporated herein byreference in its entirety. U.S. Published Application No. 20100093082and Thildeaux et al., PloSOne 5(6): e11263 (2010), each incorporatedherein by reference in its entirety, disclose use of orthoganol tRNAtechnology in vertebrete cells to produce proteins that include a pAFresidue.

In general, a prokaryote or vertebrate host cell is transformed withnucleic acid molecules encoding an orthogonal tRNA-tRNA synthetase pairwherein the tRNA is preferentially charged with an unnatural amino acid(e.g., pAcF) and the tRNA recognizes a selector codon (e.g., ambercodon). The host is then transformed with nucleic acid moleculesencoding the heavy and light chains of the antibody with one or moreselector codons at preselected locations within the nucleic acidsequence encoding the heavy or light chains. During translation of mRNAtranscribed from the nucleic acid molecules, the host cell inserts anunnatural amino acid wherever in the mRNA sequence there is a selectorcodon. For example, if the mRNA encoding the heavy chain includes oneselector codon, a heavy chain molecule will be produced that includespAcF inserted at the location encoded by the selector codon. Assembly ofthe heavy chain molecules and the light chain molecules into an antibodymolecule results in an antibody that has two pAcF residues.

Pharmaceutical Compositions

Further provided are pharmaceutical compositions, which comprise atherapeutically effective amount of one or more of the antibody peptideconjugates disclosed herein for the treatment of a metabolic disorder inan individual. Such disorders include, but are not limited to, obesity,metabolic syndrome or syndrome X, type II diabetes, complications ofdiabetes such as retinopathy, hypertension, dyslipidemias,cardiovascular disease, gallstones, osteoarthritis, and certain forms ofcancers. The obesity-related disorders herein are associated with,caused by, or result from obesity.

“Obesity” is a condition in which there is an excess of body fat. Theoperational definition of obesity is based on the Body Mass Index (BMI),calculated as body weight per height in meters squared (kg/m2).“Obesity” refers to a condition whereby an otherwise healthy subject hasa Body Mass Index (BMI) greater than or equal to 30 kg/m2, or acondition whereby a subject with at least one co-morbidity has a BMIgreater than or equal to 27 kg/m2. An “obese subject” is an otherwisehealthy subject with a Body Mass Index (BMI) greater than or equal to 30kg/m2 or a subject with at least one co-morbidity with a BMI greaterthan or equal to 27 kg/m2. A “subject at risk for obesity” is anotherwise healthy subject with a BMI of 25 kg/m2 to less than 30 kg/m2or a subject with at least one co-morbidity with a BMI of 25 kg/m2 toless than 27 kg/m2.

The increased risks associated with obesity occur at a lower Body MassIndex (BMI) in Asians. In Asian countries, including Japan, “obesity”refers to a condition whereby a subject with at least oneobesity-induced or obesity-related co-morbidity that requires weightreduction or that would be improved by weight reduction, has a BMIgreater than or equal to 25 kg/m2. In Asian countries, including Japan,an “obese subject” refers to a subject with at least one obesity-inducedor obesity-related co-morbidity that requires weight reduction or thatwould be improved by weight reduction, with a BMI greater than or equalto 25 kg/m2. In Asian countries, a “subject at risk of obesity” is asubject with a BMI of greater than 23 kg/m2 to less than 25 kg/m2.

As used herein, the term “obesity” is meant to encompass all of theabove definitions of obesity.

Obesity-induced or obesity-related co-morbidities include, but are notlimited to, diabetes, non-insulin dependent diabetes mellitus—type 2,impaired glucose tolerance, impaired fasting glucose, insulin resistancesyndrome, dyslipidemia, hypertension, hyperuricacidemia, gout, coronaryartery disease, myocardial infarction, angina pectoris, sleep apneasyndrome, Pickwickian syndrome, non-alcoholic fatty liver disease(NAFLD), non-alcoholic steatohepatitis (NASH), fatty liver; cerebralinfarction, cerebral thrombosis, transient ischemic attack, orthopedicdisorders, arthritis deformans, lumbodynia, emmeniopathy, andinfertility. In particular, co-morbidities include: hypertension,hyperlipidemia, dyslipidemia, glucose intolerance, cardiovasculardisease, sleep apnea, diabetes mellitus, and other obesity-relatedconditions.

“Treatment” (of obesity and obesity-related disorders) refers to theadministration of the compounds of the present invention to reduce ormaintain the body weight of an obese subject. One outcome of treatmentmay be reducing the body weight of an obese subject relative to thatsubject's body weight immediately before the administration of thecompounds of the present invention. Another outcome of treatment may bepreventing body weight regain of body weight previously lost as a resultof diet, exercise, or pharmacotherapy. Another outcome of treatment maybe decreasing the occurrence of and/or the severity of obesity-relateddiseases. The treatment may suitably result in a reduction in food orcalorie intake by the subject, including a reduction in total foodintake, or a reduction of intake of specific components of the diet suchas carbohydrates or fats; and/or the inhibition of nutrient absorption;and/or the inhibition of the reduction of metabolic rate; and in weightreduction in patients in need thereof. The treatment may also result inan alteration of metabolic rate, such as an increase in metabolic rate,rather than or in addition to an inhibition of the reduction ofmetabolic rate; and/or in minimization of the metabolic resistance thatnormally results from weight loss.

“Prevention” (of obesity and obesity-related disorders) refers to theadministration of the compounds of the present invention to reduce ormaintain the body weight of a subject at risk of obesity. One outcome ofprevention may be reducing the body weight of a subject at risk ofobesity relative to that subject's body weight immediately before theadministration of the compounds of the present invention. Anotheroutcome of prevention may be preventing body weight regain of bodyweight previously lost as a result of diet, exercise, orpharmacotherapy. Another outcome of prevention may be preventing obesityfrom occurring if the treatment is administered prior to the onset ofobesity in a subject at risk of obesity. Another outcome of preventionmay be decreasing the occurrence and/or severity of obesity-relateddisorders if the treatment is administered prior to the onset of obesityin a subject at risk of obesity. Moreover, if treatment is commenced inalready obese subjects, such treatment may prevent the occurrence,progression or severity of obesity-related disorders, such as, but notlimited to, arteriosclerosis, Type II diabetes, polycystic ovariandisease, cardiovascular diseases, osteoarthritis, dermatologicaldisorders, hypertension, insulin resistance, hypercholesterolemia,hypertriglyceridemia, and cholelithiasis.

The obesity-related disorders herein are associated with, caused by, orresult from obesity. Examples of obesity-related disorders includeovereating and bulimia, hypertension, diabetes, elevated plasma insulinconcentrations and insulin resistance, dyslipidemias, hyperlipidemia,endometrial, breast, prostate and colon cancer, osteoarthritis,obstructive sleep apnea, cholelithiasis, gallstones, heart disease,abnormal heart rhythms and arrythmias, myocardial infarction, congestiveheart failure, coronary heart disease, sudden death, stroke, polycysticovarian disease, craniopharyngioma, the Prader-Willi Syndrome,Frohlich's syndrome, GH-deficient subjects, normal variant shortstature, Turner's syndrome, and other pathological conditions showingreduced metabolic activity or a decrease in resting energy expenditureas a percentage of total fat-free mass, e.g, children with acutelymphoblastic leukemia. Further examples of obesity-related disordersare metabolic syndrome, also known as syndrome X, insulin resistancesyndrome, sexual and reproductive dysfunction, such as infertility,hypogonadism in males and hirsutism in females, gastrointestinalmotility disorders, such as obesity-related gastro-esophageal reflux,respiratory disorders, such as obesity-hypoventilation syndrome(Pickwickian syndrome), cardiovascular disorders, inflammation, such assystemic inflammation of the vasculature, arteriosclerosis,hypercholesterolemia, hyperuricaemia, lower back pain, gallbladderdisease, gout, and kidney cancer. The compounds of the present inventionare also useful for reducing the risk of secondary outcomes of obesity,such as reducing the risk of left ventricular hypertrophy.

The term “diabetes,” as used herein, includes both insulin-dependentdiabetes mellitus (IDDM, also known as type I diabetes) andnon-insulin-dependent diabetes mellitus (NIDDM, also known as Type IIdiabetes). Type I diabetes, or insulin-dependent diabetes, is the resultof an absolute deficiency of insulin, the hormone which regulatesglucose utilization. Type II diabetes, or insulin-independent diabetes(i.e., non-insulin-dependent diabetes mellitus), often occurs in theface of normal, or even elevated levels of insulin and appears to be theresult of the inability of tissues to respond appropriately to insulin.Most of the Type II diabetics are also obese. The compounds of thepresent invention are useful for treating both Type I and Type IIdiabetes. The compounds are especially effective for treating Type IIdiabetes. The compounds of the present invention are also useful fortreating and/or preventing gestational diabetes mellitus.

U.S. Pat. No. 6,852,690, which is incorporated herein by reference inits entirety, discloses methods for enhancing metabolism of nutrientscomprising administering to a non-diabetic patient a formulationcomprising a nutritively effective amount of one or more nutrients orany combination thereof and one or more insulinotropic peptides. Theco-agonist peptides disclosed herein are insulinotropic and can beadministered to patients with a disturbed glucose metabolism such asinsulin resistance but no overt diabetes, as well as patients who forany reason cannot receive nutrition through the alimentary canal. Suchpatients include surgery patients, comatose patients, patients in shock,patients with gastrointestinal disease, patients with digestive hormonedisease, and the like. In particular, obese patients, atheroscleroticpatients, vascular disease patients, patients with gestational diabetes,patients with liver disease such as liver cirrhosis, patients withacromegaly, patients with glucorticoid excess such as cortisol treatmentor Cushings disease, patients with activated counterregulatory hormonessuch as would occur after trauma, accidents and surgery and the like,patients with hypertriglyceridemia and patients with chronicpancreatitis can be readily and suitably nourished according to theinvention without subjecting the patient to hypo- or hyperglycemia. Inparticular, the administration to such a patient aims to provide atherapy to as rapidly as possible deliver the nutritional and caloricrequirements to the patient while maintaining his plasma glucose belowthe so-called renal threshold of about 160 to 180 milligrams perdeciliter of glucose in the blood. Although normal patients not havingglucose levels just below the renal threshold can also be treatedaccording to the invention as described above, patients with disturbedglucose metabolism such as hyperglycemic patients whose plasma glucoselevel is just above the renal threshold also find the therapy suitablefor their condition. In particular, such patients who have a degree ofhyperglycemia below the renal threshold at intermittent intervals canreceive a combination treatment of nutrients plus insulinotropicpeptides according to any of the following regimens. Normal patients notsuffering from such hyperglycemia can also be treated using the peptideanalogs disclosed herein.

The antibody peptide conjugates disclosed herein may be used in apharmaceutical composition when combined with a pharmaceuticallyacceptable carrier. Such compositions comprise atherapeutically-effective amount of one or more of the antibody peptideconjugates disclosed herein and a pharmaceutically acceptable carrier.Such a composition may also be comprised of (in addition to theco-agonist peptides disclosed herein and a carrier) diluents, fillers,salts, buffers, stabilizers, solubilizers, and other materials wellknown in the art. Compositions comprising the co-agonist peptidesdisclosed herein can be administered, if desired, in the form of saltsprovided the salts are pharmaceutically acceptable. Salts may beprepared using standard procedures known to those skilled in the art ofsynthetic organic chemistry.

The term “individual” is meant to include humans and companion ordomesticated animals such as dogs, cats, horses, and the like.Therefore, the compositions comprising a compound as disclosed hereinare also useful for treating or preventing obesity and obesity-relateddisorders in cats and dogs. As such, the term “mammal” includescompanion animals such as cats and dogs.

The term “pharmaceutically acceptable salts” refers to salts preparedfrom pharmaceutically acceptable non-toxic bases or acids includinginorganic or organic bases and inorganic or organic acids. Salts derivedfrom inorganic bases include aluminum, ammonium, calcium, copper,ferric, ferrous, lithium, magnesium, manganic salts, manganous,potassium, sodium, zinc, and the like. Particularly preferred are theammonium, calcium, magnesium, potassium, and sodium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, and basic ionexchange resins, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,histidine, hydrabamine, isopropylamine, lysine, methylglucamine,morpholine, piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine,tromethamine, and the like. The term “pharmaceutically acceptable salt”further includes all acceptable salts such as acetate, lactobionate,benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate,bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide,bromide, methylnitrate, calcium edetate, methylsulfate, camsylate,mucate, carbonate, napsylate, chloride, nitrate, clavulanate,N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate,edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate,esylate, pantothenate, fumarate, phosphate/diphosphate, gluceptate,polygalacturonate, gluconate, salicylate, glutamate, stearate,glycollylarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine,succinate, hydrobromide, tannate, hydrochloride, tartrate,hydroxynaphthoate, teoclate, iodide, tosylate, isothionate,triethiodide, lactate, panoate, valerate, and the like which can be usedas a dosage form for modifying the solubility or hydrolysischaracteristics or can be used in sustained release or pro-drugformulations. It will be understood that, as used herein, references tothe OXM analogs disclosed herein are meant to also include thepharmaceutically acceptable salts.

As utilized herein, the term “pharmaceutically acceptable” means anon-toxic material that does not interfere with the effectiveness of thebiological activity of the active ingredient(s), approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals and, more particularly, in humans. The term “carrier” refersto a diluent, adjuvant, excipient, or vehicle with which the therapeuticis administered and includes, but is not limited to such sterile liquidsas water and oils. The characteristics of the carrier will depend on theroute of administration. The antibody peptide conjugates disclosedherein may be in multimers (for example, heterodimers or homodimers) orcomplexes with itself or other peptides. As a result, pharmaceuticalcompositions of the invention may comprise one or more antibody peptideconjugates disclosed herein in such multimeric or complexed form.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially, or simultaneously.

The pharmacological composition can comprise one or more antibodypeptide conjugates disclosed herein; one or more antibody peptideconjugates disclosed herein and one or more other agents for treating ametabolic disorder; or the pharmacological composition comprising theone or more antibody peptide conjugates disclosed herein can be usedconcurrently with a pharmacological composition comprising an agent fortreating a metabolic disorder. Such disorders include, but are notlimited to, obesity, metabolic syndrome or syndrome X, type II diabetes,complications of diabetes, hypertension, dyslipidemias, cardiovasculardisease, gallstones, osteoarthritis, and certain forms of cancers.

When the pharmacological composition comprises another agent fortreating a metabolic disorder or the treatment includes a secondpharmacological composition comprising an agent for treating a metabolicdisorder, the agent includes, but are not limited to, cannabinoid (CB1)receptor antagonists, glucagon like peptide 1 (GLP-1) receptor agonists,glucagon receptor antagonists, lipase inhibitors, leptin,tetrahydrolipstatin, 2-4-dinitrophenol, acarbose, sibutramine,phentamine, fat absorption blockers, simvastatin, mevastatin, ezetimibe,atorvastatin, sitagliptin, metformin, orlistat, Qnexa, topiramate,naltrexone, bupriopion, phentermine, losartan, losartan withhydrochlorothiazide, and the like.

Examples of other active ingredients that may be administered separatelyor in the same pharmaceutical composition in combination with aco-agonist peptide as described herein include, but are not limited to:

(1) other dipeptidyl peptidase-IV (DPP-4) inhibitors (e.g., sitagliptin,alogliptin, linagliptin, vildagliptin, saxagliptin and omarigliptin);

(2) insulin sensitizers, including (i) PPARγ agonists, such as theglitazones (e.g. pioglitazone, AMG 131, MBX2044, mitoglitazone,lobeglitazone, IDR-105, rosiglitazone, and balaglitazone), and otherPPAR ligands, including (1) PPARα/γ dual agonists (e.g., ZYH2, ZYH1,GFT505, chiglitazar, muraglitazar, aleglitazar, sodelglitazar, andnaveglitazar); (2) PPARα agonists such as fenofibric acid derivatives(e.g., gemfibrozil, clofibrate, ciprofibrate, fenofibrate, bezafibrate),(3) selective PPARγ modulators (SPPARγM's), (e.g., such as thosedisclosed in WO 02/060388, WO 02/08188, WO 2004/019869, WO 2004/020409,WO 2004/020408, and WO 2004/066963); and (4) PPARγ partial agonists;(ii) biguanides, such as metformin and its pharmaceutically acceptablesalts, in particular, metformin hydrochloride, and extended-releaseformulations thereof, such as Glumetza™, Fortamet™, and GlucophageXR™;and (iii) protein tyrosine phosphatase-1B (PTP-1B) inhibitors (e.g.,ISIS-113715 and TTP814); (3) insulin or insulin analogs (e.g., insulindetemir, insulin glulisine, insulin degludec, insulin glargine, insulinlispro and inhalable formulations of each);

(4) leptin and leptin derivatives and agonists;

(5) amylin and amylin analogs (e.g., pramlintide);

(6) sulfonylurea and non-sulfonylurea insulin secretagogues (e.g.,tolbutamide, glyburide, glipizide, glimepiride, mitiglinide,meglitinides, nateglinide and repaglinide);

(7) α-glucosidase inhibitors (e.g., acarbose, voglibose and miglitol);

(8) glucagon receptor antagonists (e.g., such as those disclosed in WO98/04528, WO 99/01423, WO 00/39088, and WO 00/69810);

(9) incretin mimetics, such as GLP-1, GLP-1 analogs, derivatives, andmimetics; and GLP-1 receptor agonists (e.g., dulaglutide, semaglutide,albiglutide, exenatide, liraglutide, lixisenatide, taspoglutide,CJC-1131, and BIM-51077, including intranasal, transdermal, andonce-weekly formulations thereof);

(10) LDL cholesterol lowering agents such as (i) HMG-CoA reductaseinhibitors (e.g., simvastatin, lovastatin, pravastatin, crivastatin,fluvastatin, atorvastatin, pitavastatin and rosuvastatin), (ii) bileacid sequestering agents (e.g., colestilan, colestimide, colesevalamhydrochloride, colestipol, cholestyramine, and dialkylaminoalkylderivatives of a cross-linked dextran), (iii) inhibitors of cholesterolabsorption, (e.g., ezetimibe), and (iv) acyl CoA:cholesterolacyltransferase inhibitors, (e.g., avasimibe);

(11) HDL-raising drugs, (e.g., niacin and nicotinic acid receptoragonists, and extended-release versions thereof; MK-524A, which is acombination of niacin extended-release and the DP-1 antagonist MK-524);

(12) antiobesity compounds;

(13) agents intended for use in inflammatory conditions, such asaspirin, non-steroidal anti-inflammatory drugs or NSAIDs,glucocorticoids, and selective cyclooxygenase-2 or COX-2 inhibitors;

(14) antihypertensive agents, such as ACE inhibitors (e.g.,lisinopril,enalapril, ramipril, captopril, quinapril, and tandolapril), A-IIreceptor blockers (e.g., losartan, candesartan, irbesartan, olmesartanmedoxomil, valsartan, telmisartan, and eprosartan), renin inhibitors(e.g., aliskiren), beta blockers, and calcium channel blockers;

(15) glucokinase activators (GKAs) (e.g., AZD6370);

(16) inhibitors of 11β-hydroxysteroid dehydrogenase type 1, (e.g., suchas those disclosed in U.S. Pat. No. 6,730,690, and LY-2523199);

(17) CETP inhibitors (e.g., anacetrapib, and torcetrapib);

(18) inhibitors of fructose 1,6-bisphosphatase, (e.g., such as thosedisclosed in U.S. Pat. Nos. 6,054,587; 6,110,903; 6,284,748; 6,399,782;and 6,489,476);

(19) inhibitors of acetyl CoA carboxylase-1 or 2 (ACC1 or ACC2);

(20) AMP-activated Protein Kinase (AMPK) activators;

(21) other agonists of the G-protein-coupled receptors: (i) GPR-109,(ii) GPR-119 (e.g., MBX2982 and PSN821), and (iii) GPR-40 (e.g., TAK875,5-[4-[[(1R)-4-[6-(3-hydroxy-3-methylbutoxy)-2-methylpyridine-3-yl]-2,3-dihydro-1H-indene-1-yl]oxy]phenyl]isothiazole-3-ol1-oxide,5-(4-((3-(2,6-dimethyl-4-(3-(methylsulfonyl)propoxy)phenyl)phenyl)methoxy)phenyl)iso,5-(4-((3-(2-methyl-6-(3-hydroxypropoxy)pyridine-3-yl)-2-methylphenyl)methoxy)phenyl)isothiazole-3-ol1-oxide, and5-[4-[[3-[4-(3-aminopropoxy)-2,6-dimethylphenyl]phenyl]methoxy]phenyl]isothiazole-3-ol1-oxide);

(22) SSTR3 antagonists (e.g., such as those disclosed in WO2009/001836);

(23) neuromedin U receptor agonists (e.g., such as those disclosed in WO2009/042053, including, but not limited to, neuromedin S (NMS));

(24) SCD inhibitors;

(25) GPR-105 antagonists (e.g., such as those disclosed in WO2009/000087);

(26) SGLT inhibitors (e.g., ASP1941, SGLT-3, empagliflozin,dapagliflozin, canagliflozin, BI-10773, PF-04971729, remogloflozin,TS-071, tofogliflozin, ipragliflozin, and LX-4211);

(27) inhibitors of acyl coenzyme A:diacylglycerol acyltransferase 1 and2 (DGAT-1 and DGAT-2);

(28) inhibitors of fatty acid synthase;

(29) inhibitors of acyl coenzyme A:monoacylglycerol acyltransferase 1and 2 (MGAT-1 and MGAT-2);

(30) agonists of the TGRS receptor (also known as GPBAR1, BG37, GPCR19,GPR131, and M-BAR);

(31) ileal bile acid transporter inhibitors;

(32) PACAP, PACAP mimetics, and PACAP receptor 3 agonists;

(33) PPAR agonists;

(34) protein tyrosine phosphatase-1B (PTP-1B) inhibitors;

(35) IL-1b antibodies, (e.g., XOMA052 and canakinumab);

(36) bromocriptine mesylate and rapid-release formulations thereof, and

(37) FGF-21 and analogs and derivatives thereof;

(38) FGF21 mimetics such as agonist antibodies that binds the ß-Klothoand FCFR1c complex.

Of particular interest are metformin hydrochloride, pioglitazone,rosiglitazone, simvastatin, atorvastatin, or a sulfonylurea.

Antiobesity compounds that can be combined with the antibody peptideconjugates as disclosed herein include topiramate; zonisamide;naltrexone; phentermine; bupropion; the combination of bupropion andnaltrexone; the combination of bupropion and zonisamide; the combinationof topiramate and phentermine; fenfluramine; dexfenfluramine;sibutramine; lipase inhibitors, such as orlistat and cetilistat;melanocortin receptor agonists, in particular, melanocortin-4 receptoragonists; CCK-1 agonists; melanin-concentrating hormone (MCH) receptorantagonists; neuropeptide Y₁ or Y₅ antagonists (such as MK-0557); CB1receptor inverse agonists and antagonists (such as rimonabant andtaranabant); β₃ adrenergic receptor agonists; ghrelin antagonists;bombesin receptor agonists (such as bombesin receptor subtype-3agonists); and 5-hydroxytryptamine-2c (5-HT2c) agonists, such aslorcaserin. For a review of anti-obesity compounds that can be combinedwith compounds of the present invention, see Chaki et al., “Recentadvances in feeding suppressing agents: potential therapeutic strategyfor the treatment of obesity,” Expert Opin. Ther. Patents, 11: 1677-1692(2001); Spanswick and Lee, “Emerging antiobesity drugs,” Expert Opin.Emerging Drugs, 8: 217-237 (2003); Fernandez-Lopez, et al.,“Pharmacological Approaches for the Treatment of Obesity,” Drugs, 62:915-944 (2002); and Gadde, et al., “Combination pharmaceutical therapiesfor obesity,” Exp. Opin. Pharmacother., 10: 921-925 (2009).

In another aspect of the invention, a pharmaceutical composition isdisclosed which comprises one or more of the following agents:

(a) an antibody peptide conjugate as disclosed herein;

(b) one or more compounds selected from the group consisting of:

-   -   (1) other dipeptidyl peptidase-IV (DPP-4) inhibitors;    -   (2) insulin sensitizers, including (i) PPARγ agonists, such as        the glitazones (e.g. AMG 131, MBX2044, mitoglitazone,        lobeglitazone, IDR-105, pioglitazone, rosiglitazone, and        balaglitazone) and other PPAR ligands, including (1) PPARα/γ        dual agonists, such as ZYH1, YYH2, chiglitazar, GFT505,        muraglitazar, aleglitazar, sodelglitazar, and naveglitazar, (2)        PPARα agonists, such as fenofibric acid derivatives (e.g.,        gemfibrozil, clofibrate, ciprofibrate, fenofibrate and        bezafibrate), (3) selective PPARγ modulators (SPPARγM's),        and (4) PPARγ partial agonists; (ii) biguanides, such as        metformin and its pharmaceutically acceptable salts, in        particular, metformin hydrochloride, and extended-release        formulations thereof, such as Glumetza®, Fortamet®, and        GlucophageXR®; (iii) protein tyrosine phosphatase-1B (PTP-1B)        inhibitors, such as ISI-113715, and TTP814;    -   (3) sulfonylurea and non-sulfonylurea insulin secretagogues,        (e.g., tolbutamide, glyburide, glipizide, glimepiride,        mitiglinide, and meglitinides, such as nateglinide and        repaglinide);    -   (4) α-glucosidase inhibitors (e.g., acarbose, voglibose and        miglitol);    -   (5) glucagon receptor antagonists;    -   (6) LDL cholesterol lowering agents such as (i) HMG-CoA        reductase inhibitors (e.g., lovastatin, simvastatin,        pravastatin, cerivastatin, fluvastatin, atorvastatin,        pitavastatin, and rosuvastatin), (ii) bile acid sequestering        agents (e.g., colestilan, cholestyramine, colestimide,        colesevelam hydrochloride, colestipol, and dialkylaminoalkyl        derivatives of a cross-linked dextran), (iii) inhibitors of        cholesterol absorption, (e.g., ezetimibe), and (iv) acyl CoA:        cholesterol acyltransferase inhibitors (e.g., avasimibe);    -   (7) HDL-raising drugs, such as niacin or a salt thereof and        extended-release versions thereof; MK-524A, which is a        combination of niacin extended-release and the DP-1 antagonist        MK-524; and nicotinic acid receptor agonists;    -   (8) antiobesity compounds;    -   (9) agents intended for use in inflammatory conditions, such as        aspirin, non-steroidal anti-inflammatory drugs (NSAIDs),        glucocorticoids, and selective cyclooxygenase-2 (COX-2)        inhibitors;    -   (10) antihypertensive agents, such as ACE inhibitors (e.g.,        enalapril, lisinopril, ramipril, captopril, quinapril, and        tandolapril), A-II receptor blockers (e.g., losartan,        candesartan, irbesartan, olmesartan medoxomil, valsartan,        telmisartan, and eprosartan), renin inhibitors (e.g.,        aliskiren), beta blockers (e.g., calcium channel blockers);    -   (11) glucokinase activators (GKAs) (e.g., AZD6370);    -   (12) inhibitors of 11β-hydroxysteroid dehydrogenase type 1        (e.g., such as those disclosed in U.S. Pat. No. 6,730,690; WO        03/104207; and WO 04/058741);    -   (13) inhibitors of cholesteryl ester transfer protein (CETP),        (e.g., torcetrapib and MK-0859);    -   (14) inhibitors of fructose 1,6-bisphosphatase (e.g., such as        those disclosed in U.S. Pat. Nos. 6,054,587; 6,110,903;        6,284,748; 6,399,782; and 6,489,476);    -   (15) inhibitors of acetyl CoA carboxylase-1 or 2 (ACC1 or ACC2);    -   (16) AMP-activated Protein Kinase (AMPK) activators;    -   (17) agonists of the G-protein-coupled receptors: (i)        GPR-109, (ii) GPR-119 (e.g., MBX2982, and PSN821), and (iii)        GPR-40 (e.g., TAK875,        5-[4-[[(1R)-4-[6-(3-hydroxy-3-methylbutoxy)-2-methylpyridine-3-yl]-2,3-dihydro-1H-indene-1-yl]oxy]phenyl]isothiazole-3-ol        1-oxide,        5-(4-((3-(2,6-dimethyl-4-(3-(methylsulfonyl)propoxy)phenyl)phenyl)methoxy)phenyl)iso,        5-(4-((3-(2-methyl-6-(3-hydroxypropoxy)pyridine-3-yl)-2-methylphenyl)methoxy)phenyl)isothiazole-3-ol        1-oxide, and        5-[4-[[3-[4-(3-aminopropoxy)-2,6-dimethylphenyl]phenyl]methoxy]phenyl]isothiazole-3-ol        1-oxide);    -   (18) SSTR3 antagonists (e.g., such as those disclosed in WO        2009/011836);    -   (19) neuromedin U receptor agonists (e.g., such as those        disclosed in WO2009/042053, including, but not limited to,        neuromedin S (NMS));    -   (20) inhibitors of stearoyl-coenzyme A delta-9 desaturase (SCD);    -   (21) GPR-105 antagonists (e.g., such as those disclosed in WO        2009/000087);    -   (22) inhibitors of glucose uptake, such as sodium-glucose        transporter (SGLT) inhibitors and its various isoforms, such as        SGLT-1; SGLT-2 (e.g., ASP1941, TS071, B110773, tofogliflozin,        LX4211, canagliflozin, dapagliflozin and remogliflozin; and        SGLT-3);    -   (23) inhibitors of acyl coenzyme A:diacylglycerol        acyltransferase 1 and 2 (DGAT-1 and DGAT-2);    -   (24) inhibitors of fatty acid synthase;    -   (25) inhibitors of acyl coenzyme A:monoacylglycerol        acyltransferase 1 and 2 (MGAT-1 and MGAT-2);    -   (26) agonists of the TGRS receptor (also known as GPBAR1, BG37,        GPCR19, GPR131, and M-BAR);    -   (28) bromocriptine mesylate and rapid-release formulations        thereof, and    -   (29) IL-1b antibodies (e.g., XOMA052, and canakinumab);    -   (30) FGF-21 or analog or derivative;    -   (31) FGF21 mimetics such as agonist antibodies that binds the        ß-Klotho and FCFR1c complex; and

(c) a pharmaceutically acceptable carrier.

When an antibody peptide conjugate of the present invention is usedcontemporaneously with one or more other drugs, peptides, or proteins, apharmaceutical composition containing such other drugs, peptides, orproteins in addition to the an antibody peptide conjugate of the presentinvention may be provided. Accordingly, the pharmaceutical compositionsof the present invention include those that also contain one or moreother active ingredients, in addition to a co-agonist peptide of thepresent invention.

Methods of administrating the pharmacological compositions comprisingthe an antibody peptide conjugate disclosed herein to an individualinclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compositions can be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (for example, oral mucosa, rectaland intestinal mucosa, and the like), ocular, and the like and can beadministered together with other biologically-active agents.Administration can be systemic or local. In addition, it may beadvantageous to administer the composition into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection. Intraventricular injection may be facilitated by anintraventricular catheter attached to a reservoir (for example, anOmmaya reservoir). Pulmonary administration may also be employed by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.It may also be desirable to administer the one or more antibody peptideconjugates disclosed herein locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, topical application, by injection, bymeans of a catheter, by means of a suppository, or by means of animplant.

Various delivery systems are known and can be used to administer theantibody peptide conjugates disclosed herein including, but not limitedto, encapsulation in liposomes, microparticles, microcapsules;minicells; polymers; capsules; tablets; and the like. In one embodiment,the antibody peptide conjugates disclosed herein may be delivered in avesicle, in particular a liposome. In a liposome, the antibody peptideconjugates disclosed herein are combined, in addition to otherpharmaceutically acceptable carriers, with amphipathic agents such aslipids which exist in aggregated form as micelles, insoluble monolayers,liquid crystals, or lamellar layers in aqueous solution. Suitable lipidsfor liposomal formulation include, without limitation, monoglycerides,diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bileacids, and the like. Preparation of such liposomal formulations iswithin the level of skill in the art, as disclosed, for example, in U.S.Pat. Nos. 4,837,028 and 4,737,323. In yet another embodiment, theantibody peptide conjugates disclosed herein can be delivered in acontrolled release system including, but not limited to: a delivery pump(See, for example, Saudek, et al., New Engl. J. Med. 321: 574 (1989) anda semi-permeable polymeric material (See, for example, Howard, et al.,J. Neurosurg. 71: 105 (1989)). Additionally, the controlled releasesystem can be placed in proximity of the therapeutic target (forexample, the brain), thus requiring only a fraction of the systemicdose. See, for example, Goodson, In: Medical Applications of ControlledRelease, 1984. (CRC Press, Bocca Raton, Fla.).

The amount of the compositions comprising one or more of the antibodypeptide conjugates disclosed herein which will be effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder or condition, and may be determined by standardclinical techniques by those of average skill within the art. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theoverall seriousness of the disease or disorder, and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances. Ultimately, the attending physician will decide theamount of the composition with which to treat each individual patient.Initially, the attending physician will administer low doses of thecomposition and observe the patient's response. Larger doses of thecomposition may be administered until the optimal therapeutic effect isobtained for the patient, and at that point the dosage is not increasedfurther. In general, the daily dose range lie within the range of fromabout 0.001 mg to about 100 mg per kg body weight of a mammal,preferably 0.01 mg to about 50 mg per kg, and most preferably 0.1 to 10mg per kg, in single or divided doses. On the other hand, it may benecessary to use dosages outside these limits in some cases. However,suitable dosage ranges for intravenous administration of thecompositions comprising the one or more antibody peptide conjugatesdisclosed herein are generally about 5-500 micrograms (μg) of activecompound per kilogram (Kg) body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient. Ultimately the attending physician will decide on theappropriate duration of therapy using compositions comprising the one ormore co-agonist peptides disclosed herein of the present invention.Dosage will also vary according to the age, weight and response of theindividual patient.

Further provided is a pharmaceutical pack or kit, comprising one or morecontainers filled with one or more of the ingredients of thepharmaceutical compositions and/or antibody peptide conjugates disclosedherein. Optionally associated with such container(s) may be a notice inthe form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, which noticereflects approval by the agency of manufacture, use or sale for humanadministration.

The following examples are intended to promote a further understandingof the present invention.

Example 1

The peptides shown in Table 1 were synthesized by solid phase usingFmoc/t-Bu chemistry on a peptide multisynthesizer Symphony (ProteinTechnologies Inc.) on a Rink-amide PEG-PS resin, Champion (BiosearchTechnologies, 150 μmol scale, loading 0.28 mmol/g) All the amino acidswere dissolved at a 0.3 M concentration in DMF. The acylation reactionswere performed for 45 min with 5-fold excess of activated amino acidover the resin free amino groups. The amino acids were activated withequimolar amounts of HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) solution 0.3 M in DMF,and a 2-fold molar excess of DIEA (N,N-diisopropylethylamine), solution2M in NMP.

The side chain protecting groups were: tert-butyl for Ser, D-Ser, Thrand Tyr; trityl for Asn, Gln and His; tert-butoxy-carbonyl for Lys, Trp;and, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl for Arg; TheN-terminal residue was acylated as Boc-His(Trt)-OH. To produce sequenceshaving the C-terminal Gln-OH, sequence ID 14-21, 47-54 and 60-61, theRink-amide PEG-PS resin was acylated with Fmoc-Glu-OtBu activated withequimolar amounts of HATU(O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate) solution 0.3 M in DMF, and a 2-fold molar excess ofDIEA (N,N-diisopropylethylamine), solution 2M in NMP. The lysinederivatized with the spacer residue and aminoxy functionality isincorporated either as Fmoc-Lys(Alloc)-OH (Alloc=allyloxycarbonyl) orFmoc-Lys(Dde)-OH,Dde=[1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl]. The followingno natural aminoacids: Fmoc-β-(3-benzothienyl)-alanine, Fmoc-L-7Azatryptophan, Fmoc-L-2-naphthylalanine, Fmoc-L-(3-Pyridyl)alanine,Fmoc-L-methionine sulfoxide, Fmoc-L-homoleucine,Fmoc-L-homocycloexylalanine, Fmoc-L-3-(2quinoyl)-alanine,Fmoc-L-4,4′-biphenylalanine, Fmoc-L-N-methyl-tryptophane,Fmoc-L-hexafluoroleucine, Fmoc-L-5-hydroxy tryptophan,Fmoc-L-alpha-methylphenylalanine, Fmoc-L-alpha-methyl-leucine,Fmoc-L-alpha-methyltryptophan, Fmoc-L-β-homotryptophan whereincorporated by manual coupling, using HOAt (Hydroxybenzoazatriazole)and DIC (N,N′-diisopropylcarbodiimide) as activators. At the end of thepeptide assembly on solid phase, the Alloc protecting group was removedfrom either Lys31 or Lys24 or Lys10 with Pd(PPh₃)₄ and PhSiH₃, while theDde protecting group was removed by treatment with a solution 2% ofNH₂NH₂ in DMF. The side chain derivatization was performed by manualcoupling of Bis-Boc aminoxy acetic acid or the appropriate PEG spacerresidue and the Bis-Boc aminoxy acetic acid using HOAt and DIC asactivators (4 fold excess for 4 hours). The amino PEG spacers are shownin Table 2.

TABLE 2 Spacers Spacer NO: Name 1 PEG2

2 PEG4

3 PEG6

4 PEG8

5 PEG24

6 PEG36

For SEQ ID NO.52, 53, and 54, the Lys at position 24 was protected bythe orthogonal Alloc (allyloxycarbonyl) group, while the Lys at position10 was protected with Dde[1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl] group. At the end ofthe peptide assembly on solid phase, the Dde protecting group wasremoved by treatment with a solution 2% of NH₂NH₂ in DMF and 200 molarexcess of allyl alcohol and derivatization was performed by manualcoupling of two Fmoc-Glu-OtBu residues and palmitic acid, activated withDIC and HOAt. After Lys10 derivatization, the Alloc protecting group wasremoved from Lys24 with Pd(PPh₃)₄ and PhSiH₃ and the side chain wasassembled by manual coupling of Bis-Boc aminoxyacetic acid (SEQ ID NO.51), Fmoc-N-amido-dPEG₂-acid and Bis-Boc aminoxyacetic acid (SEQ ID NO.52), Fmoc-N-amido-dPEG₄-acid and Bis-Boc aminoxyacetic acid (SEQ ID NO.54), activated with DIC and HOAt.

For SEQ ID NO:47, 48, 49, 50, and 51, Lys10 was derivatized with Bis-Bocaminoxyacetic acid, Fmoc-Glu-OtBu and Bis-Boc aminoxyacetic acid, twoFmoc-Glu-OtBu residues and Bis-Boc aminoxyacetic acid,Fmoc-N-amido-dPEG₂-acid and Bis-Boc aminoxyacetic acid,Fmoc-N-amido-dPEG₄-acid and Bis-Boc aminoxyacetic acid respectively. ForSEQ ID NO.24, E24 and K28 were incorporated as Fmoc-L-glutamic acid5-allyl ester and Fmoc-Lys(Alloc)-OH respectively, while K31 wasorthogonally protected with ivDde[Fmoc-N^(ε)-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine].The Alloc protective group was removed as previously described andlactam bridge was performed between E24 and K28, using Pyclock[(6-Chlorobenzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate], HOAt and DIPEA as activators. After lactamformation, the ivDde group on K31 was removed by treatment with asolution 2% of NH₂NH₂ in DMF and the side chain was assembled by manualcoupling of Fmoc-N-amido-dPEG4-acid and Bis-Boc aminoxyacetic acid,activated with DIC and HOAt.

For various sequences, the following amino acid analogs wereincorporated into the peptide at position 25 or 27.

At the end of the synthesis, the dry peptide-resins were individuallytreated with 25 mL of the cleavage mixture, 88% TFA, 5% phenol, 2%triisopropylsilane and 5% water for 1.5 hours at room temperature. Eachresin was filtered and the volume of the solution was reduced then addedto cold methyl-t-butyl ether with 10% of aminoxy acetic acid. Aftercentrifugation, the peptide pellets were washed with fresh coldmethyl-t-butyl ether containing 10% of aminoxy acetic acid. The processwas repeated twice. Final pellets were dried, resuspended in H₂O, 20%acetonitrile, and lyophilized.

The crude peptides (140 mg in 3 ml of DMSO) were purified byreverse-phase HPLC using preparative Waters)(Bridge C18 (50×150 mm, 5μm, 100 Å) and using as eluents (A) 0.1% TFA in water and (B) 0.1% TFAin acetonitrile.

Analytical HPLC was performed on a Acquity UPLC Waters Chromatographwith a BEH130 C18 or BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm, at 45°C., using H2O, 0.1% TFA (A) and CH3CN, 0.1% TFA (B) as solvents. Thepeptides were characterized by electrospray mass spectrometry on anAcquity SQ Detector.

Analytical Characterization of Peptides:

The purified peptide SEQ ID NO:1 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MH+found: 3722.7 Da; Mw expected: 3723.09 Da).

The purified peptide SEQ ID NO:2 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H2O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on an Acquity SQ Detector. (MWfound: 3867.0 Da; MW expected: 3868.25 Da).

The purified peptide SEQ ID NO:3 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solvents.The peptides were characterized by electrospray mass spectrometry on anAcquity SQ Detector (MW found: 3970.5 Da; MW expected: 3970.38).

The purified peptide SEQ ID NO:4 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4058.1 Da; MW expected: 4058.48 Da).

The purified peptide SEQ ID NO:5 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4146.6 Da; MW expected: 4146.59 Da).

The purified peptide SEQ ID NO:6 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4852.8 Da; MW expected: 4851.53 Da).

The purified peptide SEQ ID NO:7 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 5381.6 Da; MW expected: 5380.06 Da).

The purified peptide SEQ ID NO:8 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3957.2 Da; Mw expected: 3957.34 Da).

The purified peptide SEQ ID NO:9 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3869.7 Da; MW expected: 3870.22 Da).

The purified peptide SEQ ID NO:10 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3972.6 Da; MW expected: 3972.35 Da).

The purified peptide SEQ ID NO:11 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4060.2 Da; MW expected: 4060.46 Da).

The purified peptide SEQ ID NO:12 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4148.4 Da; Mw expected: 4147.4 Da).

The purified peptide SEQ ID NO:13 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4854.0 Da; Mw expected: 4853.4 Da).

The purified peptide SEQ ID NO:14 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3723.0 Da; MW expected: 3724.07 Da).

The purified peptide SEQ ID NO:15 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3868.8 Da; MW expected: 3869.23 Da).

The purified peptide SEQ ID NO:16 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solvents.The peptide was characterized by electrospray mass spectrometry on anAcquity SQ Detector. (MW found: 3970.8 Da; MW expected: 3971.36 Da).

The purified peptide SEQ ID NO:17 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4059.6 Da; MW expected: 4059.47 Da).

The purified peptide SEQ ID NO:18 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4146.6 Da; MW expected: 4147.57 Da).

The purified peptide SEQ ID NO:19 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3898.5 Da; MW expected: 3898.35 Da).

The purified peptide SEQ ID NO:20 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3871.9 Da; MW expected: 3871.2 Da).

The purified peptide SEQ ID NO:21 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3972.0 Da; MW expected: 3973.33 Da).

The purified peptide SEQ ID NO:22 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3613.8 Da; MW expected: 3613.92 Da).

The purified peptide SEQ ID NO:23 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: Da; MW expected: 3716.05 Da).

The purified peptide SEQ ID NO:24 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3967.5 Da; MW expected: 3966.43 Da).

The purified peptide SEQ ID NO:25 was characterized on an Acquity UPLCWaters Chromatograph, with BEH 300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3988.2 Da; MW expected: 3987.43 Da).

The purified peptide SEQ ID NO:26 was characterized on an Acquity UPLCWaters Chromatograph, with BEH 300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3971.1 Da; MW expected: 3971.37 Da).

The purified peptide SEQ ID NO:27 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3981.9 Da; MW expected: 3981.4 Da).

The purified peptide SEQ ID NO:28 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3932.7 Da; MW expected: 3932.33 Da).

The purified peptide SEQ ID NO:29 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3954.6 Da; MW expected: 3954.38 Da).

The purified peptide SEQ ID NO:30 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3987.0 Da; MW expected: 3986.38 Da).

The purified peptide SEQ ID NO:31 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3966.6 Da; MW expected: 3966.35 Da).

The purified peptide SEQ ID NO:32 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4007.1 Da; MW expected: 4006.43 Da).

The purified peptide SEQ ID NO:33 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3983.7 Da; MW expected: 3982.39 Da).

The purified peptide SEQ ID NO:134 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4008.6 Da; MW expected: 4007.4 Da).

The purified peptide SEQ ID NO:35 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3984.1 Da; MW expected: 3984.4 Da).

The purified peptide SEQ ID NO:36 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3943.2 Da; MW expected: 3943.31 Da).

The purified peptide SEQ ID NO:37 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4079.4 Da; MW expected: 4079.3 Da).

The purified peptide SEQ ID NO:38 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3987.0 Da; MW expected: 3987.36 Da).

The purified peptide SEQ ID NO:39 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3913.5 Da; MW expected: 3913.32 Da).

The purified peptide SEQ ID NO:40 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3912.9 Da; MW expected: 3913.28 Da).

The purified peptide SEQ ID NO:41 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3899.7 Da; MW expected: 3898.27 Da).

The purified peptide SEQ ID NO:42 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3946.2 Da; MW expected: 3946.3 Da).

The purified peptide SEQ ID NO:43 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3869.7 Da; MW expected: 3870.26 Da).

The purified peptide SEQ ID NO:44 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3911.7 Da; MW expected: 3912.34 Da).

The purified peptide SEQ ID NO:45 was characterized on an Acquity UPLCWaters Chromatograph, with BEH300 C4 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3984.5 Da; MW expected: 3985.39 Da).

The purified peptide SEQ ID NO:46 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3689.1 Da; MW expected: 3689.03 Da).

The purified peptide SEQ ID NO:47 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3819.0 Da; MW expected: 3818.14 Da).

The purified peptide SEQ ID NO:48 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3947.4 Da; MW expected: 3947.26 Da).

The purified peptide SEQ ID NO:49 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3834.6 Da; MW expected: 3834.19 Da).

The purified peptide SEQ ID NO:50 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 3936.3 Da; MW expected: 3936.32 Da).

The purified peptide SEQ ID NO:51 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4184.7 Da; MW expected: 4185.7 Da).

The purified peptide SEQ ID NO:52 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4330.8 Da; MW expected: 4330.87 Da).

The purified peptide SEQ ID NO:53 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H₂O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solventsand by electrospray mass spectrometry on a Acquity SQ Detector. (MWfound: 4431.6 Da; MW expected: 4433.06 Da).

The purified peptide SEQ ID NO:54 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H2O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solvents.The peptides were characterized by electrospray mass spectrometry on aAcquity SQ Detector (MW found: 3974 Da; MW expected: 3970.38 Da).

The purified peptide SEQ ID NO:55 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H2O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solvents.The peptides were characterized by electrospray mass spectrometry on aAcquity SQ Detector (MW found: 3974.6 Da; MW expected: 3974.32 Da).

The purified peptide SEQ ID NO:56 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H2O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solvents.The peptides were characterized by electrospray mass spectrometry on aAcquity SQ Detector (MW found: 4777.6 Da; MW expected: 4778.49 Da).

The purified peptide SEQ ID NO:57 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H2O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solvents.The peptides were characterized by electrospray mass spectrometry on aAcquity SQ Detector (MW found: 4734.4 Da; MW expected: 4735.42 Da).

The purified peptide SEQ ID NO:58 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H2O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solvents.The peptides were characterized by electrospray mass spectrometry on aAcquity SQ Detector (MW found: 4777.3 Da; MW expected: 4778.49 Da).

The purified peptide SEQ ID NO:59 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H2O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solvents.The peptides were characterized by electrospray mass spectrometry on aAcquity SQ Detector (MW found: 4734.3 Da; MW expected: 4735.42 Da).

The purified peptide SEQ ID NO:60 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H2O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solvents.The peptides were characterized by electrospray mass spectrometry on aAcquity SQ Detector (MW found: 4778.5 Da; MW expected: 4779.47 Da).

The purified peptide SEQ ID NO:61 was characterized on an Acquity UPLCWaters Chromatograph, with BEH130 C18 Acquity Waters 2.1×100 mm, 1.7 μm,at 45° C., using H2O, 0.1% TFA (A) and CH₃CN, 0.1% TFA (B) as solvents.The peptides were characterized by electrospray mass spectrometry on aAcquity SQ Detector (MW found: 4735.3 Da; MW expected: 4736.4 Da).

Example 2

Site specific conjugation using oxime chemistry was used to make theantibody peptide conjugates. The antibodies comprising para-acetylphenylalanine (pAcF) were produced using

The antibodies comprising pAcF were purified from clarified cell culturemedia by affinity chromatograph (AbSolute protein A, NovaSep) followedby cation exchange chromatography (SP 650S, Tosoh Biosciences).

The antibodies comprising pAcF were buffer exchanged into 50 mM sodiumacetate; 200 mM glycine; 2.5% trehalose; 0-20% DMSO, pH 4.0-4.5 andconcentrated to 1-20 mg/mL. 60 mM acetic hydrazide and 10-15 molarequivalents of peptide comprising aminooxy were added and reacted for16-72 hours at 28-30° C. The reaction was monitored by LCMS. Theantibody conjugates (APCs) were purified over a SP 650S column (TosohBiosciences) to remove excess reagents. The conjugates were bufferexchanged into 50 mM Histidine; 100 mM NaCl; 2.5% Trehalose; pH 6.0,0.22 μm filtered, and stored at 4° C.

Conjugation Analysis.

Conjugation efficiencies and drug to antibody ratio (DAR) values weredetermined by LC-MS intact mass. The APCs were applied over a PLRP-S, 8μm, 1000 Å column, 2.1×150 mm (Agilent) at 80° C. and eluted with alinear gradient from 25% B to 55% B over 20 minutes (A: Water, 0.05%TFA; B: Acetonitrile, 0.04% TFA). The mass spectra were obtained on anAgilent 6510 Q-Tof mass spectrometer with MassHunter software anddeconvoluted using BioConfirm. DAR values were determined with relativepeak abundances of the masses correlating to conjugated antibody.

Additionally, APCs were also analyzed by size-exclusion chromatographyfor monomer content. Purified APCs were applied onto a Tosoh G3000SWx1 5μm column, 7.8×300 mm (Tosoh) using 0.2M potassium phosphate, 0.25Mpotassium chloride, 10% IPA pH 6.0 as the mobile phase. Target monomercontent for the APCs was >95%.

Activity Measurement for APCs (cAMP)

APCs were received as aqueous solutions formulated in 50 mM L-histidine,100 mM NaCl, 2.5% trehalose, pH 6.0. They were serially diluted in thesame formulation buffer to generate 10 point titrations. The APCsolutions were then transferred into 384-well assay plates (150 nl/well)using Echo (Labcyte). Assay ready frozen cells expressing human GLP-1R,human GCGR, mouse GLP1R and mouse GCGR were suspended in growth mediaconsisting of DMEM medium (GIBCO), 10% FBS (GIBCO), 1×NEAA(GIBCO), 1×P/S (GIBCO), 200 ug/ml Hygromycin (GIBCO) and 10 ug/ml Blasticidin(GIBCO). Cells were then diluted in assay buffer consisting of PBS(GIBCO), 7.5% BSA (Perkin Elmer), 100 uM RO 20-1724 (Sigma), with orwithout 20% human (MP Biomedical) or mouse serum (Bioreclamation). Thecell suspensions (15 ul) were then added to the assay plates containingthe peptide solutions (30,000 cells/well for human GCGR, mouse GCGR,mouse GLP1R and 10,000 cells/well for human GLP1R). The assay plateswere incubated for 1 hour at room temperature in the dark. Production ofcAMP was determined using HitHunter™ cAMPXS kit (DiscoverX) followingmanufacturer protocol. The plates were incubated overnight at roomtemperature in the dark. Luminescence was measured using an EnVisionMultilabel plate reader (Perkin Elmer). Native GLP-1 and Glucagon(Bachem) are used as control peptides. EC50 values were calculated usinga 4 parameter logistic fit based on the Levenberg-Marquardt algorithm.Results are shown in Table 3.

TABLE 3 Measured Activities Human Human GCGR GLP1R Human 20% Human 20%pAcF Peptide GCGR serum GLP1R serum mAb position* Number (nM) (nM) (nM)(nM) Ambody HS32 4702 IA IA ~31 ~39 Ambody HS32 4704 1.67 2.98 4.6 2.8Ambody HS32 4739 4.4 6.6 6.7 6.5 Ambody HS32 4740 3.8 4 3.8 5.7 AmbodyHS32 4840 4.6 7.2 8.4 8.2 Ambody HS32 4841 2.9 ~4.1 8.8 7.1 Ambody HS324842 4.5 4.9 7 6.2 Ambody HS32 4953 0.33 1.20 0.40 0.74 Ambody HS32 50093.1 3.6 3.5 5.3 Ambody HS32 5009 ~3.1 ~3.6 ~3.5 ~5.3 Ambody HS32 5010~3.7 ~3.9 ~4.3 6.1 Ambody HS32 5048 3.10 3.4 7.1 7.4 Ambody HS32 50493.6 4.2 9.8 8.6 Ambody HS32 5050 4.4 4.0 10.5 8.5 Ambody HS32 5051 4.54.4 11 9.9 Ambody HS32 5052 1.6 1.8 16 13 Ambody HS32 5053 2.4 3.8 10.68.4 Ambody HS32 5058 2.5 2.8 1.7 2.3 Ambody HS32 5059 3.9 4.0 4.0 3.3Ambody HS32 5275 ~90 >100 5.7 5.2 Ambody HS32 5307 2.4 3.5 6.5 5.4Ambody HS32 5314 4.0 6.5 18.4 11.4 Ambody HS32 5420 3.7 7.2 9.7 8.8Ambody HS32 5615 3.7 5.1 5.3 4.6 Ambody HS32 6115 2.64 4.05 3.34 3.36Ambody HQ16 4740 7.2 8.0 2.7 3.3 Ambody HG33 4740 4.2 5.0 3.4 3.2 AmbodyHD56 4740 5.6 9.1 1.9 2.2 Ambody HA114 4740 14.3 13.9 4.3 5.0 AmbodyLK125 4740 54.8 65.9 9.4 10.4 Ambody LE142 4740 23.3 33.5 5.3 5.8 AmbodyHS179 4740 40.5 49.4 7.6 7.7 Ambody HT198 4740 70.6 40 23.9 25.1 AmbodyHN211 4740 28.8 31.3 7.9 7.1 Ambody HD56 4740 10.61 10.39 4.56 3.36Ambody HA114 4740 10 10.00 4.83 4.06 Ambody LE142 4740 36 38.66 7.777.07 Ambody HT198 5615 3.66 5.06 5.27 4.59 Ambody LK125 5615 7.35 9.955.10 5.41 Ambody HQ16 5615 4.16 5.64 6.58 5.51 Ambody HG33 4740 2.563.83 4.67 4.03 Ambody HA114 4740 9.07 11.50 5.50 5.06 Ambody LE142 56157.64 7.80 5.18 5.30 Ambody LK125 5615 4.79 8.50 6.43 6.60 Ambody HS325759 5.27 5.15 4.92 4.47 Ambody HS32 5760 3.22 3.44 3.62 2.85 AmbodyHS32 5763 5.70 6.01 4.56 3.94 Ambody HS32 5764 6.23 5.38 8.19 5.40Ambody HS32 5765 6.71 7.03 18.59 5.50 Ambody HS32 5761 2.85 4.83 5.314.92 Ambody HS32 5762 2.40 3.74 4.55 4.29 Ambody HS32 5762 2.40 3.744.55 4.29 Ambody HS32 5766 6.30 7.84 5.40 4.99 Ambody HS32 5799 2.865.44 5.25 4.70 Ambody HS32 5798 5.94 5.54 4.84 5.59 Ambody HS32 58663.50 4.32 4.82 4.39 Ambody HS32 6052 2.42 3.50 5.73 5.11 Ambody HS326053 3.99 4.57 4.73 4.13 Ambody HS32 6062 3.73 4.04 3.53 3.53 AmbodyHS32 6376 9.86 10.4 6.73 6.27 Ambody HS32 6377 27.00 27.2 19.34 15.84Ambody HS32 6378 6.01 6.5 8.06 6.61 Ambody HS32 6379 6.82 7.6 5.75 5.64Ambody HS32 6380 3.56 3.99 4.03 3.17 Ambody HS32 6381 6.48 5.41 4.843.95 Ambody HS32 6382 4.10 4.15 4.27 3.54 Ambody HS32 6383 5.30 5.044.91 4.06 Ambody HS32 6384 3.40 3.90 4.69 4.18 Ambody HS32 6385 2.402.37 3.08 2.52 Ambody HS32 6541 25.06 16.36 5.63 5.17 Ambody HS32 6542200.00 >200 5.51 4.64 Ambody HS32 6543 7.34 8.73 5.30 4.64 Ambody HS326544 200.00 >200 4.56 4.27 Ambody HS32 6545 5.59 5.49 3.65 4.02 AmbodyHS32 6546 105.90 91.11 4.18 3.86 IA = inactive *pAcF position - H or Lis for heavy chain or light chain, followed by the amino acid replacedby the pAcF and its position in the amino acid sequenceIn Vivo Efficacy Model

Male diet-induced obese (DIO) mice, which at 4-week-old were fed withhigh fat (60% kcal) diet D12492 to induce obesity. Mice were singlyhoused, acclimated and given free access to D12492 and water. Mice weredivided into groups of sixteen mice per group based on initial averagebody weight. Each group of mice was intravenously injected once at day 0with a dose of Antibody peptide conjugate (APC). The APCs studied wereAmbody-HS32pAcF-Pep4740 @ 3 mg/kg, Ambody-HS32pAcF-Pep4704 @ 3 and 10mg/kg, Ambody-HS32pAcF-Pep5615 @ 3 and 10 mg/kg. The administered dosesused 50 Mm Histidine, 100 Mm NaCl, pH 6.0, 2.5% Trehalose as formulationbuffer to make the doses. Body weight, food intake and were measureddaily during treatment.

The in vivo effects of Ambody-HS32pAcF-Pep4704, Ambody-HS32pAcF-Pep5615of the invention were tested in diet-induced obese (DIO) mice that weremaintained on a high fat diet for 20 weeks and had an initial bodyweight of about 48 grams. Mice were administered intravenously once atday 0. The APCs in this study included Ambody-HS32pAcF-Pep4740 at a doseof 3 mg/kg as a positive control. The Ambody-HS32pAcF-Pep4704 andAmbody-HS32pAcF-Pep5615 were administered at doses of 3 and 10 mg/kg,respectively. Cumulative body weight change (grams) was measured eachday of the study except on day 3. Results are shown in FIG. 2 and areexpressed as mean±SEM.

The Ambody-HS32pAcF-Pep5615 (Ambody-P5615) tested in this studydemonstrated a significant (p<0.0001 vs Ambody-HS32pAcF-Pep4740(Ambody-P4740), 1 way ANOVA) weight loss over the course of the studycompared to the Ambody-HS32pAcF-Pep4740 treated groups of mice. On day10 of the study, mice that were administered 3 mg/kg of theAmbody-HS32pAcF-Pep5615 (Ambody-P5615) exhibited an approximate weightloss of 5 grams, while a dose of 10 mg/kg of the Ambody-HS32pAcF-Pep5615(Ambody-P5615) led to a body weight loss of 10.3 grams. Mice that wereadministered 3 mg/kg of the Ambody-HS32pAcF-Pep4740 (Ambody-P4740)exhibited weight loss of 0.92 grams. Mice that were administered 3 mg/kgand 10 mg/kg of the Ambody-HS32pAcF-Pep4704 (Ambody-P4704) exhibitedapproximately 0.55 grams and 1.0 grams of weight loss, respectively.

Body weight change is expressed as percent relative toAmbody-HS32pAcF-Pep4740 (FIG. 3). Mice administered a dose of 3 mg/kgthe Ambody-HS32pAcF-Pep4740 (Ambody-P4740) exhibited approximately 1.9%weight loss at day 10 compared to mice administered with a dose of 3mg/kg and 10 mg/kg the Ambody-HS32pAcF-Pep5615(Ambody-P5615), whoexhibited weight loss of approximately 10.5% or 21.5% respectively. Miceadministered with a dose of 3 mg/kg the Ambody-HS32pAcF-Pep4704(Ambody-P4704) exhibited weight loss of approximately 1.1%, while miceadministered with a dose 10 mg/kg the Ambody-HS32pAcF-Pep4704(Ambody-P4704) exhibited weight loss of approximately 2.0%.

As shown in FIG. 4, DIO mice that were administered withAmbody-HS32pAcF-Pep4704, exhibited a significant less reduction incumulative food intake after the first 3 days of dosing compare toAmbody-HS32pAcF-Pep4740 (Ambody-P4740) (**p<0.005 ***p<0.001****p<0.0001vs Pep4740, 1way ANOVA, Newman-Keuls multiple comparisons). Mice thatwere administered with Ambody-HS32pAcF-Pep5615 (Ambody-P5615) exhibiteda significant suppression of food intake at first 3 days. On day 4 to 7,there were no significant changes in food intake except the miceadministered Ambody-HS32pAcF-Pep5615 (Ambody-P5615) at the 10 mg/kg. Day8 through day 10, there were no changes in food intake.

In Vivo Plasma Exposure of APC Constructs

In vivo plasma exposure was monitored during the efficacy evaluation ofAPCs in DIO mice (mouse model and efficacy evaluation described under‘In vivo efficacy model section’). All groups received an intravenoussingle bolus dose at day 0 of APC. The APCs studied wereAmbody-HS32pAcF-Pep4740 @ 3 mg/kg, Ambody-HS32pAcF-Pep4704 @ 3 and 10mg/kg, Ambody-HS32pAcF-Pep5615 @ 3 and 10 mg/kg. The administered dosesused 50 Mm Histidine, 100 Mm NaCl, pH 6.0, 2.5% Trehalose as formulationbuffer to make the doses. Whole blood was collected on Day 1, 3, 7 and10 in test tubes pretreated with dipotassium-EDTA and processed toplasma by centrifugation (4000 g, 2-8° C.). Plasma concentration wasmeasured by conjugate immunoassay as described below.

Quantitation of Circulating APC Compounds in Mouse Serum (ConjugateImmunoassay)

A two site sandwich immunoassay was set up to quantitate the APCs on theMeso Scale Discovery (MSD) assay platform (Meso Scale Discovery). MSDStreptavidin Gold Multi-Array 96-Well Plate was blocked with 150 μL of5% BSA in PBS and incubated overnight at 4° C. at 50 rpm shaking. Theplate was washed 3 times using 200 μL of Wash Buffer (PBS with 0.05%Tween 20) and was coated with capture antibody (Merck & Co., Inc.,Biotinylated Mouse×[GCG_H] mAb, TC140.20F1.C1 IgG2b/Kappa) using avolume of 25 μL per well at 2 μg/mL in Assay Buffer (0.5% BSA [wt/v],0.05% Tween 20 [v/v], 0.25% CHAPS [wt/v], 5 mM EDTA in PBS) andincubated for 1 hour at room temperature at 600 rpm. The plate waswashed 3 times with Wash Buffer and 25 μL of MSD Diluent 4 was addedinto each well. Then 25 μL of calibrators, controls, and samples wereadded into designated wells. The APC calibrators were made for each APCas an eight-point curve starting at 500 ng/mL and by serially diluting1:4 to 0.12 ng/mL at point 7. Calibrator point 8 was 2% mouse plasma inAssay Buffer for assay background. The serum samples were diluted at aminimum of 1:50 with Assay Buffer. The plate was then placed on a plateshaker for 2 hours at room temperature at 600 rpm. Following a wash stepas described before, 25 μL of the detector antibody (Mouse anti-Hu IgG4,Southern Biotech Clone HP6025, conjugated with MSD ST-label rutheniumtris-bipyridine chelate according to manufacturer's protocol) was addedat a concentration of 1 μg/mL in Assay Buffer. Following a one hourincubation at room temperature at 600 rpm, the plate was washed asdescribed above and processed in MSD Meso Sector S 600 instrument tomeasure electrochemiluminescence signal according to manufacturer'sprotocol.

FIG. 5 demonstrates the impact of spacer design on circulatory stabilityof the conjugated APC. At comparable doses, Ambody-HS32pAcF-Pep5615(Ambody-5615) had the highest plasma exposure followed byAmbody-HS32pAcF-Pep4740 (Ambody-4740) and then Ambody-HS32pAcF-Pep4704(Ambody-4704). The results show that by increasing the length of thespacer for linking the peptide to the antibody, the serum half-life ofthe peptide could be extended or enhanced.

TABLE 4 ANTIBODY SEQUENCES SEQ ID NO: Description Amino Acid Sequence 64Palivizumab DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMEIWYQQKPGKAPKLL Light ChainIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 65 AntibodyQVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKAL Heavy ChainEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLG66 Ambody QVTLRESGPALVKPTQTLTLTCTFSGFSLSTXGMSVGWIRQPPGKAL Heavy ChainEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTAT HS32YYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTSES X at 32 = pAcF TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLG67 Ambody QVTLRESGPALVKPTXTLTLTCTFSGFSLSTSGMSVGWIRQPPGKAL Heavy ChainEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTAT HQ16YYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTSES X at 16 = pAcFTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLG68 Ambody QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSXMSVGWIRQPPGKAL Heavy ChainEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTAT HG33YYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTSES X at 33 = pAcFTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLG69 Ambody QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKAL Heavy ChainEWLADIWWXDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTAT HD56YYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTSES X at 56 = pAcFTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLG70 Ambody QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKAL Heavy ChainEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTAT HT114YYCARSMITNWYFDVWGAGXTVTVSSASTKGPSVFPLAPCSRSTSES X at 114 = pAcFTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLG71 Ambody DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLI Light ChainYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYP LK125FTFGGGTKLEIKRTVAAPSVFIFPPSDEQLXSGTASVVCLLNNFYPR X at 125 = pAcFEAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 72 AmbodyDIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLI Light ChainYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYP LE142FTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR X at 142 = pAcFXAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 73 AmbodyQVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKAL Heavy ChainEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTAT HS179YYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTSES X at 179 = pAcFTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQXSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLG74 Ambody QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKAL Heavy ChainEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTAT HT198YYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTSES X at 198 = pAcFTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGXKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLG75 Ambody QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKAL Heavy ChainEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTAT HN211YYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTSES X at 211 = pAcFTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSXTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLG

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

What is claimed:
 1. An antibody peptide conjugate comprising: anantibody having a light chain having the amino acid sequence of SEQ IDNO:64 and a heavy chain having the amino acid sequence of SEQ ID NO:65conjugated to a peptide having the amino acid sequence (SEQ ID NO: 62)His-Xaa²-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Xaa¹⁶-Arg-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asp-Thr-Lys-Gln

wherein Xaa² is α-aminoisobutyric acid (aib) or D-Ser; Xaa¹⁶ is aib; atleast one of the amino acids at position 10, 24, or 31 is substitutedwith a Lys or the amino acids at positions 10 and 24 are eachsubstituted with a Lys; and optionally the peptide comprises up to threeadditional amino acid substitutions; wherein the heavy chain includes asubstitution of the amino acid at position 16, 32, 33, 56, 114, 179,198, or 211 with a para-acetylphenylalanine (pAcF) or the light chainincludes a substitution of the amino acid at position 125 or 142 withpAcF; wherein the peptide comprises either (i) an aminooxy acid residuecovalently linked to the epsilon amino group of the Lys at position 10,24, or 31 or (ii) an aminooxy acid residue covalently linked to theepsilon amino group of the Lys at position 10, 24, or 31 via apolyethylene glycol (PEG) spacer, a γGlu spacer, or a γGlu-γGlu spacer;wherein the pAcF residue is covalently linked to the aminooxy acidresidue; and wherein the antibody peptide conjugate is an agonist of theglucagon receptor and the glucagon-like peptide 1 receptor.
 2. Theantibody peptide conjugate of claim 1, wherein the peptide comprises afatty acid covalently linked to the epsilon amino group of the Lys atposition 10 via a γGlu-γGlu spacer.
 3. The antibody peptide conjugate ofclaim 2, wherein the fatty acid comprises a C14, C15, C16, C17, C18,C19, or C20 fatty acid.
 4. The antibody peptide conjugate of claim 3,wherein the fatty acid comprises the C14 fatty acid.
 5. The antibodypeptide conjugate of claim 1, comprising the polyethylene glycol spacerthat comprises 2, 4, 6, 8, 24, or 36 ethoxy units.
 6. The antibodypeptide conjugate of claim 1 wherein: the antibody has a light chainhaving the amino acid sequence of SEQ ID NO:64, SEQ ID NO:71, or SEQ IDNO:72 and a heavy chain having the amino acid sequence of SEQ ID NO:65,SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70,SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75 conjugated to a peptidecomprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, or SEQ IDNO:61, wherein the aminooxy acid residue of the peptide is covalentlylinked to the paracetylphenylalanine (pAcF) residue of the light chainor heavy chain, with the proviso that if the light chain has the aminoacid sequence of SEQ ID NO:64 then heavy chain does not have the aminoacid sequence of SEQ ID NO:65 and if the light chain has the amino acidsequence of SEQ ID NO:71 or 72 then the heavy chain has the amino acidsequence of SEQ ID NO:65.
 7. A pharmaceutical composition comprising theantibody peptide conjugate of claim 1 and a pharmaceutically acceptablecarrier.
 8. A method for treating a metabolic disease or disorder in apatient, comprising administering to a patient in need thepharmaceutical composition of claim 1 to treat the metabolic disease ordisorder.
 9. The method of claim 8, wherein the metabolic disease ordisorder comprises diabetes, non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), or obesity.
 10. The method ofclaim 9, wherein the diabetes comprises Type 1 diabetes, Type IIdiabetes, or gestational diabetes.